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Here, we demonstrate that single strand annealing (SSA) can be co-opted for the precise autocatalytic excision of a drive element. We have termed this technology Repeat Mediated Excision of a Drive Element (ReMEDE). By engineering direct repeats flanking the drive allele and inducing a double-strand DNA break (DSB) at a second endonuclease target site within the allele, we increased the utilization of SSA repair. ReMEDE was incorporated into the mutagenic chain reaction (MCR) gene drive targeting the yellow gene of Drosophila melanogaster, successfully replacing drive alleles with wild-type alleles. Sequencing across the Cas9 target site confirmed transgene excision by SSA after pair-mated outcrosses with yReMEDE females, revealing ~4% inheritance of an engineered silent TcG marker sequence. However, phenotypically wild-type flies with alleles of indeterminate biogenesis also were observed, retaining the TGG sequence (~16%) or harboring a silent gGG mutation (~0.5%) at the PAM site. Additionally, ~14% of alleles in the F2 flies were intact or uncut paternally inherited alleles, indicating limited maternal deposition of Cas9 RNP. Although ReMEDE requires further research and development, the technology has some promising features as a gene drive mitigation strategy, notably its potential to restore wild-type populations without additional transgenic releases or large-scale environmental modifications. Author summary: CRISPR/Cas9-based autonomous homing gene drives have the potential to drastically reduce or eliminate vector-borne diseases, agricultural pests, and invasive species. However, their use raises significant regulatory, ethical, environmental, and sociopolitical concerns, particularly regarding unintended consequences. Responsible application of this powerful technology necessitates the development of control measures to halt or reverse any undesired outcomes. Several mitigation strategies have been proposed and, in some cases, demonstrated in laboratory settings, but their effectiveness in addressing concerns surrounding the use of gene drives in nature still remains uncertain. Therefore, ongoing investigation and consideration of additional control strategies is prudent. This manuscript describes proof-of-concept studies for a novel gene drive mitigation strategy, which we have termed Repeat Mediated Excision of a Drive Element (ReMEDE). While further research and development is required prior to any possible application of this strategy, the ReMEDE concept offers a theoretical potential to restore transgene-free populations with wild-type phenotypes after a well-defined period of efficient gene drive. [ABSTRACT FROM AUTHOR]

Benzyl butyl phthalate (BBP), a plasticizer found in a wide range of consumer products including vinyl flooring, carpet backing, food packaging, personal care products, and children's toys, is an endocrine-disrupting chemical linked to impaired reproduction and development in humans. Despite evidence that BBP exposure perturbs the integrity of male and female gametes, its direct effect on early meiotic events is understudied. Here, using the nematode Caenorhabditis elegans, we show that BBP exposure elicits a non-monotonic dose response on the rate of X-chromosome nondisjunction measured using a high-throughput screening platform. From among the range of doses tested (1, 10, 100 and 500 μM BBP), we found that 10 μM BBP elicited the strongest effect on the germline, resulting in increased germ cell apoptosis and chromosome organization defects. Mass spectrometry analysis shows that C. elegans efficiently metabolizes BBP into its primary metabolites, monobutyl phthalate (MBP) and monobenzyl phthalate (MBzP), and that the levels of BBP, MBP, and MBzP detected in the worm are within the range detected in human biological samples. Exposure to 10 μM BBP leads to germlines with enlarged mitotic nuclei, altered meiotic progression, activation of a p53/CEP-1-dependent DNA damage checkpoint, increased double-strand break levels throughout the germline, chromosome morphology defects in oocytes at diakinesis, and increased oxidative stress. RNA sequencing analysis indicates that BBP exposure results in the altered expression of genes involved in xenobiotic metabolic processes, extracellular matrix organization, oocyte morphogenesis, meiotic cell cycle, and oxidoreduction. Taken together, we propose that C. elegans exposure to BBP leads to increased oxidative stress and double-strand break formation, thereby compromising germline genomic integrity and chromosome segregation. Author summary: Endocrine disrupting chemicals (EDCs) have been linked to various health problems including effects on reproductive health. Benzyl butyl phthalate (BBP) is a commonly used plasticizer found in many consumer products that has been shown to act as an EDC and affect human reproduction and development. However, how it might impact the germline and affect the specialized cell division program of meiosis, which results in the formation of haploid gametes (i.e. eggs and sperm), remained unclear. Here, using the nematode C. elegans, we show that BBP exposure at levels within the range of what is detected in humans impairs accurate chromosome segregation, which can result in aneuploidy. Similar to mammals, BBP exposure in C. elegans results in a non-monotonic dose response, whereby lower doses result in the strongest effects, and BBP is efficiently metabolized, underscoring the use of this model system for gaining mechanistic insights into how this EDC impacts the germline. BBP results in increased levels of DNA double-strand breaks (DSBs), activation of a DNA damage checkpoint, and altered chromosome morphology in exposed germlines. We propose that BBP exposure alters gene expression and results in increased oxidative stress, resulting in increased DSBs, thereby compromising chromosome morphology and accurate segregation. [ABSTRACT FROM AUTHOR]

The DNA damage response (DDR) constitutes a vital cellular process that safeguards genome integrity. This biological process involves substantial alterations in chromatin structure, commonly orchestrated by epigenetic enzymes. Here, we show that the epigenetic modifier N-terminal acetyltransferase 4 (Nat4), known to acetylate the alpha-amino group of serine 1 on histones H4 and H2A, is implicated in the response to DNA damage in S. cerevisiae. Initially, we demonstrate that yeast cells lacking Nat4 have an increased sensitivity to DNA damage and accumulate more DNA breaks than wild-type cells. Accordingly, upon DNA damage, NAT4 gene expression is elevated, and the enzyme is specifically recruited at double-strand breaks. Delving deeper into its effects on the DNA damage signaling cascade, nat4-deleted cells exhibit lower levels of the damage-induced modification H2AS129ph (γH2A), accompanied by diminished binding of the checkpoint control protein Rad9 surrounding the double-strand break. Consistently, Mec1 kinase recruitment at double-strand breaks, critical for H2AS129ph deposition and Rad9 retention, is significantly impaired in nat4Δ cells. Consequently, Mec1-dependent phosphorylation of downstream effector kinase Rad53, indicative of DNA damage checkpoint activation, is reduced. Importantly, we found that the effects of Nat4 in regulating the checkpoint signaling cascade are mediated by its N-terminal acetyltransferase activity targeted specifically towards histone H4. Overall, this study points towards a novel functional link between histone N-terminal acetyltransferase Nat4 and the DDR, associating a new histone-modifying activity in the maintenance of genome integrity. Author summary: Chromatin structure alterations are central for cells to efficiently respond to DNA damage since DNA assaults do not simply occur on 'naked' DNA but in the context of chromatin. Cells have developed an integral process to mitigate DNA damage, known as the DNA damage response (DDR), which involves instrumental chromatin dynamic changes often regulated by histone-modifying enzymes. Our work demonstrates that N-terminal acetyltransferase Nat4 regulates the response to DNA damage. Importantly, cells lacking Nat4 are more susceptible to genotoxic-induced DNA damage, as well as accumulate higher number of DNA breaks. Accordingly, Nat4 expression is induced by DNA damage and specifically localizes at DNA double-strand breaks. In agreement with this, the absence of Nat4 impairs the DDR signaling, since the distribution of DNA damage-induced phosphorylation on serine 129 of histone H2A and the subsequent binding of Rad9 surrounding the break are robustly reduced. Both events are regulated by Mec1 kinase, whose recruitment at the DNA double-strand break is significantly decreased when Nat4 is absent. Consistently, downstream activation of the DNA damage checkpoint, indicative by Rad53 phosphorylation, is significantly reduced. Finally, this work supports that Nat4 functions in DNA damage response through its N-terminal acetyltransferase activity, specifically towards histone H4. Collectively, our data reveal a novel molecular and biological role for Nat4 in the response to DNA damage, and thus implicating a new player in genome integrity. [ABSTRACT FROM AUTHOR]

We report here the successful labelling of meiotic prophase I DNA synthesis in the flowering plant, Arabidopsis thaliana. Incorporation of the thymidine analogue, EdU, enables visualisation of the footprints of recombinational repair of programmed meiotic DNA double-strand breaks (DSB), with ~400 discrete, SPO11-dependent, EdU-labelled chromosomal foci clearly visible at pachytene and later stages of meiosis. This number equates well with previous estimations of 200–300 DNA double-strand breaks per meiosis in Arabidopsis, confirming the power of this approach to detect the repair of most or all SPO11-dependent meiotic DSB repair events. The chromosomal distribution of these DNA-synthesis foci accords with that of early recombination markers and MLH1, which marks Class I crossover sites. Approximately 10 inter-homologue cross-overs (CO) have been shown to occur in each Arabidopsis male meiosis and, athough very probably under-estimated, an equivalent number of inter-homologue gene conversions (GC) have been described. Thus, at least 90% of meiotic recombination events, and very probably more, have not previously been accessible for analysis. Visual examination of the patterns of the foci on the synapsed pachytene chromosomes corresponds well with expectations from the different mechanisms of meiotic recombination and notably, no evidence for long Break-Induced Replication DNA synthesis tracts was found. Labelling of meiotic prophase I, SPO11-dependent DNA synthesis holds great promise for further understanding of the molecular mechanisms of meiotic recombination, at the heart of reproduction and evolution of eukaryotes. Author summary: Sexual reproduction involves the fusion of two cells, one from each parent. To maintain a stable chromosome complement across generations, these specialized reproductive cells must be produced through a specialized cell division called meiosis. Meiosis halves the chromosome complement of gametes and recombines the parental genetic contributions in each gamete, generating the genetic variation that drives evolution. The complex mechanisms of meiotic recombination have been intensely studied for many years and we now know that it involves the repair of programmed chromosomal breaks through recombination with intact template DNA sequences on another chromatid. At the molecular level, this is known to involve new DNA synthesis at the sites of repair/recombination and we report here the successful identification and characterisation of this DNA neo-synthesis during meiosis in the flowering plant Arabidopsis. Both the characteristics and numbers of these DNA synthesis tracts accord with expectations from theory and earlier studies. Potentially applicable to studies in many organisms, this approach provides indelible footprints in the chromosomes and has the great advantage of freeing researchers from dependence on indirect methods involving detection of proteins involved in these dynamic processes. [ABSTRACT FROM AUTHOR]

Ribosomal DNA (rDNA), which encodes ribosomal RNA, is an essential but unstable genomic element due to its tandemly repeated nature. rDNA's repetitive nature causes spontaneous intrachromatid recombination, leading to copy number (CN) reduction, which must be counteracted by a mechanism that recovers CN to sustain cells' viability. Akin to telomere maintenance, rDNA maintenance is particularly important in cell types that proliferate for an extended time period, most notably in the germline that passes the genome through generations. In Drosophila, the process of rDNA CN recovery, known as 'rDNA magnification', has been studied extensively. rDNA magnification is mediated by unequal sister chromatid exchange (USCE), which generates a sister chromatid that gains the rDNA CN by stealing copies from its sister. However, much remains elusive regarding how germ cells sense rDNA CN to decide when to initiate magnification, and how germ cells balance between the need to generate DNA double-strand breaks (DSBs) to trigger USCE vs. avoiding harmful DSBs. Recently, we identified an rDNA-binding Zinc-finger protein Indra as a factor required for rDNA magnification, however, the underlying mechanism of action remains unknown. Here we show that Indra is a negative regulator of rDNA magnification, balancing the need of rDNA magnification and repression of dangerous DSBs. Mechanistically, we show that Indra is a repressor of RNA polymerase II (Pol II)-dependent transcription of rDNA: Under low rDNA CN conditions, Indra protein amount is downregulated, leading to Pol II-mediated transcription of rDNA. This results in the expression of rDNA-specific retrotransposon, R2, which we have shown to facilitate rDNA magnification via generation of DBSs at rDNA. We propose that differential use of Pol I and Pol II plays a critical role in regulating rDNA CN expansion only when it is necessary. Author summary: Ribosomal DNA (rDNA) exists as tandemly-repeated copies in eukaryotic genome, making it unstable due to spontaneous intrachromatid recombination that causes copy number loss. The germline, which is the sole cell type that transmits genome from generation to generation, must expand rDNA copy number to counteract spontaneous copy number loss. It has been shown that the process of rDNA copy number expansion, called rDNA magnification, involves rDNA binding protein Indra and retrotransposon R2. However, the underlying molecular mechanism of rDNA magnification and how Indra and R2 may functionally intersect remained unknown. This study shows that Indra is a transcriptional repressor of the intergenic spacer (IGS) sequence of rDNA, orchestrating R2 expression and rDNA copy number expression. Low rDNA copy number downregulates Indra, leading to expression of IGS, which in turn leads to expression of R2. It was found that IGS expression is mediated by RNA polymerase II, which is specifically recruited to nucleolus upon rDNA copy number reduction. Together, the results lead to a model how rDNA copy number reduction triggers the process of rDNA copy number expansion. [ABSTRACT FROM AUTHOR]

Accurate repair of DNA double-strand breaks (DSBs) is essential for the maintenance of genome integrity, as failure to repair DSBs can result in cell death. The cell has evolved two main mechanisms for DSB repair: non-homologous end-joining (NHEJ) and homology-directed repair (HDR), which includes single-strand annealing (SSA) and homologous recombination (HR). While certain factors like age and state of the chromatin are known to influence DSB repair pathway choice, the roles of developmental stage, tissue type, and sex have yet to be elucidated in multicellular organisms. To examine the influence of these factors, DSB repair in various embryonic developmental stages, larva, and adult tissues in Drosophila melanogaster was analyzed through molecular analysis of the DR-white assay using Tracking across Indels by DEcomposition (TIDE). The proportion of HR repair was highest in tissues that maintain the canonical (G1/S/G2/M) cell cycle and suppressed in both terminally differentiated and polyploid tissues. To determine the impact of sex on repair pathway choice, repair in different tissues in both males and females was analyzed. When molecularly examining tissues containing mostly somatic cells, males and females demonstrated similar proportions of HR and NHEJ. However, when DSB repair was analyzed in male and female premeiotic germline cells utilizing phenotypic analysis of the DR-white assay, there was a significant decrease in HR in females compared to males. This study describes the impact of development, tissue-specific cycling profile, and, in some cases, sex on DSB repair outcomes, underscoring the complexity of repair in multicellular organisms. Author summary: DNA double-strand breaks (DSBs) are toxic lesions that threaten genome integrity. If not repaired accurately and efficiently, this damage may lead to cell death or tumorigenesis. Cells are able to repair DSBs by two major pathways: non-homologous end joining (NHEJ) and homologous recombination (HR). While factors such as cell cycle phase and age impact the repair pathway choice between NHEJ and HR, understanding the implications of repair in multicellular organisms, which contain heterogeneous tissues at different developmental stages is less clear. Furthermore, the impact of sex on repair in different tissues also remains to be elucidated. We have investigated these factors using an established DSB repair assay to measure repair outcomes in various tissues, developmental stages, and both sexes in Drosophila melanogaster. We found that repair by HR is favored in tissues and developmental stages that contain cells exhibiting the canonical cell cycle. While this was consistent in both males and females in most tissues, females suppress HR repair in their premeiotic germline. Our results demonstrate the complexity of repair pathway choice in multicellular organisms and underscores the impact of sex on DSB repair. [ABSTRACT FROM AUTHOR]

Meiotic recombination between homologous chromosomes is initiated by the formation of hundreds of programmed double-strand breaks (DSBs). Approximately 10% of these DSBs result in crossovers (COs), sites of physical DNA exchange between homologs that are critical to correct chromosome segregation. Virtually all COs are formed by coordinated efforts of the MSH4/MSH5 and MLH1/MLH3 heterodimers, the latter representing the defining marks of CO sites. The regulation of CO number and position is poorly understood, but undoubtedly requires the coordinated action of multiple repair pathways. In a previous report, we found gene-trap disruption of the DNA helicase, FANCJ (BRIP1/BACH1), elicited elevated numbers of MLH1 foci and chiasmata. In somatic cells, FANCJ interacts with numerous DNA repair proteins including MLH1, and we hypothesized that FANCJ functions with MLH1 to regulate the major CO pathway. To further elucidate the meiotic function of FANCJ, we produced three new Fancj mutant mouse lines via CRISPR/Cas9 gene editing: a full-gene deletion, truncation of the N-terminal Helicase domain, and a C-terminal dual-tagged allele. We also generated an antibody against the C-terminus of the mouse FANCJ protein. Surprisingly, none of our Fancj mutants show any change in either MLH1 focus counts during pachynema or total CO number at diakinesis of prophase I. We find evidence that FANCJ and MLH1 do not interact in meiosis; further, FANCJ does not co-localize with MSH4, MLH1, or MLH3 in meiosis. Instead, FANCJ co-localizes with BRCA1 and TOPBP1, forming discrete foci along the chromosome cores beginning in early meiotic prophase I and densely localized to unsynapsed chromosome axes in late zygonema and to the XY chromosomes in early pachynema. Fancj mutants also exhibit a subtle persistence of DSBs in pachynema. Collectively, these data indicate a role for FANCJ in early DSB repair, but they rule out a role for FANCJ in MLH1-mediated CO events. Author summary: Errors during meiotic chromosome segregation can result in aneuploidy in offspring, a leading cause of birth defects, pregnancy loss, and infertility. Meiotic crossovers (COs), exchanges of genetic material between parental chromosomes, are critical to ensuring faithful segregation. Nearly all COs are formed by the mismatch repair proteins, MSH4/MSH5 and MLH1/MLH3. Although the number of COs is highly controlled, the mechanisms underpinning this regulation remain poorly understood. We previously found that disruption of the DNA Helicase, FANCJ, caused an increase in CO number. In somatic cells, FANCJ is known to function in numerous DNA repair pathways and interact with diverse repair proteins, including MLH1. Thus, we hypothesized FANCJ functions with MLH1 to regulate meiotic CO formation. In this study we report that, surprisingly, FANCJ does not interact with MLH1 in mouse meiosis. Instead, FANCJ appears earlier during meiosis and does not spatially coincide with MLH1 or MSH4. Further, loss of FANCJ has no impact on CO number, but does cause an increase of unrepaired DNA breaks in late meiotic prophase. These results indicate that FANCJ is dispensable for CO formation, but likely functions in early DNA repair processes that are nonetheless important for maintaining germ cell genomic integrity. [ABSTRACT FROM AUTHOR]

With the release of the telomere-to-telomere human genome sequence and the availability of both long-read sequencing and optical genome mapping techniques, the identification of copy number variants (CNVs) and other structural variants is providing new insights into human genetic disease. Different mechanisms have been proposed to account for the novel junctions in these complex architectures, including aberrant forms of DNA replication, non-allelic homologous recombination, and various pathways that repair DNA breaks. Here, we have focused on a set of structural variants that include an inverted segment and propose that they share a common initiating event: an inverted triplication with long, unstable palindromic junctions. The secondary rearrangement of these palindromes gives rise to the various forms of inverted structural variants. We postulate that this same mechanism (ODIRA: origin-dependent inverted-repeat amplification) that creates the inverted CNVs in inherited syndromes also generates the palindromes found in cancers. [ABSTRACT FROM AUTHOR]

Telomeric repeat-containing RNA (TERRA) is a class of long noncoding RNAs (lncRNAs) that are transcribed from subtelomeric to telomeric region of chromosome ends. TERRA is prone to form R-loop structures at telomeres by invading into telomeric DNA. Excessive telomere R-loops result in telomere instability, so the TERRA level needs to be delicately modulated. However, the molecular mechanisms and factors controlling TERRA level are still largely unknown. In this study, we report that the RNA binding protein RBMX is a novel regulator of TERRA level and telomere integrity. The expression level of TERRA is significantly elevated in RBMX depleted cells, leading to enhanced telomere R-loop formation, replication stress, and telomere instability. We also found that RBMX binds to TERRA and the nuclear exosome targeting protein ZCCHC8 simultaneously, and that TERRA degradation slows down upon RBMX depletion, implying that RBMX promotes TERRA degradation by regulating its transportation to the nuclear exosome, which decays nuclear RNAs. Altogether, these findings uncover a new role of RBMX in TERRA expression regulation and telomere integrity maintenance, and raising RBMX as a potential target of cancer therapy. Author summary: Telomeres are repetitive DNA: protein structures that protect linear chromosome ends from being recognized as double-strand DNA breaks. Telomeres bear very high loads of replication stress, and are essential for chromosomal stability and genome stability. Telomeric repeat-containing RNA (TERRA) is transcribed from subtelomeres to telomeres, and is prone to form R-loop structures by invading into telomeric DNA. In telomerase-negative human cancer cell lines, which maintain telomere length by homologous recombination-based alternative lengthening of telomeres (ALT) mechanism, both TERRA levels and telomeric R-loops are upregulated. Excessive telomere R-loops result in telomere instability. Therefore, only limited number of TERRA is required for telomere homeostasis. Here we show that, the RNA binding motif protein encoded on the X chromosome (RBMX), also known as hnRNP G, is a novel regulator of TERRA level and telomere integrity. Our data point to a completely new role of RBMX in TERRA expression regulation and telomere integrity maintenance, and open up a potential way for cancer therapy. In addition, this study also raises the possibility that RBMX also targeted other nuclear RNA to nuclear exosome for degradation. [ABSTRACT FROM AUTHOR]

Single nucleotide variants are the most frequent type of sequence changes detected in the genome and these are frequently variants of uncertain significance (VUS). VUS are changes in DNA for which disease risk association is unknown. Thus, methods that classify the functional impact of a VUS can be used as evidence for variant interpretation. In the case of the breast and ovarian cancer specific tumor suppressor protein, BRCA1, pathogenic missense variants frequently score as loss of function in an assay for homology-directed repair (HDR) of DNA double-strand breaks. We previously published functional results using a multiplexed assay for 1056 amino acid substitutions residues 2–192 in the amino terminus of BRCA1. In this study, we have re-assessed the data from this multiplexed assay using an improved analysis pipeline. These new analysis methods yield functional scores for more variants in the first 192 amino acids of BRCA1, plus we report new results for BRCA1 amino acid residues 193–302. We now present the functional classification of 2172 BRCA1 variants in BRCA1 residues 2–302 using the multiplexed HDR assay. Comparison of the functional determinations of the missense variants with clinically known benign or pathogenic variants indicated 93% sensitivity and 100% specificity for this assay. The results from BRCA1 variants tested in this assay are a resource for clinical geneticists for evidence to evaluate VUS in BRCA1. Author summary: Most missense substitutions in BRCA1 are variants of unknown significance (VUS), and individuals with a VUS in BRCA1 cannot know from genetic information alone whether this variant predisposes to breast or ovarian cancer. We apply a multiplexed functional assay for homology directed repair of DNA double strand breaks to assess variant impact on this important BRCA1 protein function. We analyzed 2172 variants in the amino-terminus of BRCA1 and demonstrate that variants that are known as pathogenic have a loss of function in the DNA repair assay. Conversely, variants that are known to be benign are functionally normal in the multiplexed assay. We suggest that these functional determinations of BRCA1 variants can be used to augment the information that clinical cancer geneticists provide to patients who have a VUS in BRCA1. [ABSTRACT FROM AUTHOR]

Chromosome movements and licensing of synapsis must be tightly regulated during early meiosis to ensure accurate chromosome segregation and avoid aneuploidy, although how these steps are coordinated is not fully understood. Here we show that GRAS-1, the worm homolog of mammalian GRASP/Tamalin and CYTIP, coordinates early meiotic events with cytoskeletal forces outside the nucleus. GRAS-1 localizes close to the nuclear envelope (NE) in early prophase I and interacts with NE and cytoskeleton proteins. Delayed homologous chromosome pairing, synaptonemal complex (SC) assembly, and DNA double-strand break repair progression are partially rescued by the expression of human CYTIP in gras-1 mutants, supporting functional conservation. However, Tamalin, Cytip double knockout mice do not exhibit obvious fertility or meiotic defects, suggesting evolutionary differences between mammals. gras-1 mutants show accelerated chromosome movement during early prophase I, implicating GRAS-1 in regulating chromosome dynamics. GRAS-1-mediated regulation of chromosome movement is DHC-1-dependent, placing it acting within the LINC-controlled pathway, and depends on GRAS-1 phosphorylation at a C-terminal S/T cluster. We propose that GRAS-1 coordinates the early steps of homology search and licensing of SC assembly by regulating the pace of chromosome movement in early prophase I. Author summary: Successful sexual reproduction depends on the formation of gametes (i.e., eggs and sperm) carrying a correct number of chromosomes. This involves a series of well-choreographed steps during meiosis, the specialized cell division program resulting in the formation of eggs and sperm. Some of these steps include pairing between homologous chromosomes, assembly of a scaffold (the synaptonemal complex) between homologs, and inter-homolog recombination. These steps must be properly coordinated to ensure normal meiotic progression. Here, we show that the GRAS-1 protein in the nematode C. elegans, which shares sequence conservation with the mammalian proteins GRASP/Tamalin and CYTIP, contributes to normal meiotic progression by coordinating early events in meiosis. GRAS-1 is required for the normal speed of chromosome movements early in prophase I in a manner dependent of its phosphorylation and on dynein heavy chain. Delayed homologous chromosome pairing, synaptonemal complex assembly, and DNA double-strand break repair progression are partially rescued by the expression of human CYTIP in gras-1 mutants, supporting functional conservation. We identified a new factor coordinating the early steps of pairing and synapsis by regulating the pace of chromosome movement in early prophase I. [ABSTRACT FROM AUTHOR]

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder caused by progressive loss of motor neurons and there is currently no effective therapy. Cytoplasmic mislocalization and aggregation of TAR DNA-binding protein 43 kDa (TDP-43) within the CNS is a pathological hallmark in sporadic ALS and prion-like propagation of pathogenic TDP-43 is thought to be implicated in disease progression. However, cell-to-cell transmission of pathogenic TDP-43 in the human CNS has not been confirmed experimentally. Here we used induced pluripotent stem cells (iPSCs)-derived cerebral organoids as recipient CNS tissue model that are anatomically relevant human brain. We injected postmortem spinal cord protein extracts individually from three non-ALS or five sporadic ALS patients containing pathogenic TDP-43 into the cerebral organoids to validate the templated propagation and spreading of TDP-43 pathology in human CNS tissue. We first demonstrated that the administration of spinal cord extracts from an ALS patient induced the formation of TDP-43 pathology that progressively spread in a time-dependent manner in cerebral organoids, suggesting that pathogenic TDP-43 from ALS functioned as seeds and propagated cell-to-cell to form de novo TDP-43 pathology. We also reported that the administration of ALS patient-derived protein extracts caused astrocyte proliferation to form astrogliosis in cerebral organoids, reproducing the pathological feature seen in ALS. Moreover, we showed pathogenic TDP-43 induced cellular apoptosis and that TDP-43 pathology correlated with genomic damage due to DNA double-strand breaks. Thus, our results provide evidence that patient-derived pathogenic TDP-43 can mimic the prion-like propagation of TDP-43 pathology in human CNS tissue. Our findings indicate that our assays with human cerebral organoids that replicate ALS pathophysiology have a promising strategy for creating readouts that could be used in future drug discovery efforts against ALS. Author summary: Amyotrophic lateral sclerosis (ALS) is a disease characterized by the degeneration of selected groups of neuronal cells. Mislocalization of the TAR DNA-binding protein 43 kDa (TDP-43) from the nucleus of neuronal cells toward the cytoplasm is a pathological hallmark of most cases of ALS. Owing to its structure TDP-43 has a high aggregation propensity when outside the nucleus. Here we used a a form of self-organizing three-dimensional cell culture model (aka cerebral organoid) to test if TDP-43 could contribute the progression of the disease that occurs across the central nervous system of patients. TDP-43 is hypothesized to contribute to the progression of the disease by inducing the propagation of its aggregated form across cells. Protein extracts prepared from postmortem spinal cord of ALS patients showing aggregated TDP-43 were separately injected into cerebral organoids derived from an ALS case and from a control individual. This approach demonstrated the spreading of aggregated TDP-43 across the cells of the injected organoids derived from an ALS case; by comparison, the same spreading was very limited within organoids from the control individual. Critically, protein extracts prepared from non-diseased individuals did not result in the propagation of aggregated TDP-43. Our finding could be used to evaluate treatments aiming to prevent the propagation of aggregated TDP-43 established to be pathogenic. [ABSTRACT FROM AUTHOR]

Although homologous recombination between transposable elements can drive genomic evolution in yeast by facilitating chromosomal rearrangements, the details of the underlying mechanisms are not fully clarified. In the genome of the yeast Saccharomyces cerevisiae, the most common class of transposon is the retrotransposon Ty1. Here, we explored how Cas9-induced double-strand breaks (DSBs) directed to Ty1 elements produce genomic alterations in this yeast species. Following Cas9 induction, we observed a significant elevation of chromosome rearrangements such as deletions, duplications and translocations. In addition, we found elevated rates of mitotic recombination, resulting in loss of heterozygosity. Using Southern analysis coupled with short- and long-read DNA sequencing, we revealed important features of recombination induced in retrotransposons. Almost all of the chromosomal rearrangements reflect the repair of DSBs at Ty1 elements by non-allelic homologous recombination; clustered Ty elements were hotspots for chromosome rearrangements. In contrast, a large proportion (about three-fourths) of the allelic mitotic recombination events have breakpoints in unique sequences. Our analysis suggests that some of the latter events reflect extensive processing of the broken ends produced in the Ty element that extend into unique sequences resulting in break-induced replication. Finally, we found that haploid and diploid strain have different preferences for the pathways used to repair double-stranded DNA breaks. Our findings demonstrate the importance of DNA lesions in retrotransposons in driving genome evolution. Author summary: Transposable elements can affect gene expression and gene function. Recombination between elements located on the same chromosome can produce deletions, duplications, and inversions, whereas recombination between transposable elements on non-homologous chromosomes can generate translocations. Thus, recombination between transposable elements is an important cause of genetic variation. In this report, we present a comprehensive analysis of recombination events induced in both haploid and diploid strains of Saccharomyces cerevisiae by targeting the Ty1 family of retrotransposons with CRISPR/Cas9. Chromosome rearrangements were stimulated more than 1000-fold. DNA sequencing, using both conventional short "reads" and ultra-long "reads", showed that almost all of these rearrangements had Ty1 elements at their breakpoints. In contrast, we also observed a stimulation of allelic mitotic recombination events between homologs, most of which did not involve direct Ty-Ty interactions. Our results provided novel insights into the mechanism of genome evolution as regulated by repetitive sequences. [ABSTRACT FROM AUTHOR]

Programmed DNA double-strand break (DSB) formation is essential for achieving accurate chromosome segregation during meiosis. DSB repair timing and template choice are tightly regulated. However, little is known about how DSB distribution and the choice of repair pathway are regulated along the length of chromosomes, which has direct effects on the recombination landscape and chromosome remodeling at late prophase I. Here, we use the spatiotemporal resolution of meiosis in the Caenorhabditis elegans germline along with genetic approaches to study distribution of DSB processing and its regulation. High-resolution imaging of computationally straightened chromosomes immunostained for the RAD-51 recombinase marking DSB repair sites reveals that the pattern of RAD-51 foci throughout pachytene resembles crossover distribution in wild type. Specifically, RAD-51 foci occur primarily along the gene-poor distal thirds of the chromosomes in both early and late pachytene, and on both the X and the autosomes. However, this biased off-center distribution can be abrogated by the formation of excess DSBs. Reduced condensin function, but not an increase in total physical axial length, results in a homogeneous distribution of RAD-51 foci, whereas regulation of H3K9 methylation is required for the enrichment of RAD-51 at off-center positions. Finally, the DSB recognition heterodimer cKU-70/80, but not the non-homologous end-joining canonical ligase LIG-4, contributes to the enriched off-center distribution of RAD-51 foci. Taken together, our data supports a model by which regulation of the chromatin landscape, DSB levels, and DSB detection by cKU-70/80 collaborate to promote DSB processing by homologous recombination at off-center regions of the chromosomes in C. elegans. Author summary: Accurate chromosome segregation during meiosis is essential for all sexually reproducing organisms. Errors during meiosis can result in infertility, stillbirths, miscarriages, and birth defects. Therefore, understanding how different steps of meiosis are regulated is of vital importance to reproductive health. One such key step is the formation of programmed DNA double-strand breaks (DSBs), which are required for recombination between homologous chromosomes. DSBs are a source of genetic variability but also of genome instability; thus, regulating their formation and correct repair is paramount. In the present study, we identify different factors regulating the distribution of meiotic DSB repair sites marked by the RAD-51 recombinase along the chromosomes in the nematode Caenorhabditis elegans. We find that either excess levels of breaks or alterations in chromosome compaction can produce a homogeneous distribution of RAD-51 foci along the chromosome length. Moreover, we find that biased RAD-51 foci distribution to the terminal thirds of the chromosomes in wild type worms is regulated by the epigenetic landscape and DNA repair proteins. Our study reveals a variety of factors involved in the complex regulation of DSB distribution and repair by homologous recombination during meiosis in C. elegans. [ABSTRACT FROM AUTHOR]

During meiosis, recombination between homologous chromosomes (homologs) generates crossovers that promote proper segregation at the first meiotic division. Recombination is initiated by Spo11-catalyzed DNA double strand breaks (DSBs). 5' end resection of the DSBs creates 3' single strand tails that two recombinases, Rad51 and Dmc1, bind to form presynaptic filaments that search for homology, mediate strand invasion and generate displacement loops (D-loops). D-loop processing then forms crossover and non-crossover recombinants. Meiotic recombination occurs in two temporally distinct phases. During Phase 1, Rad51 is inhibited and Dmc1 mediates the interhomolog recombination that promotes homolog synapsis. In Phase 2, Rad51 becomes active and functions with Rad54 to repair residual DSBs, making increasing use of sister chromatids. The transition from Phase 1 to Phase 2 is controlled by the meiotic recombination checkpoint through the meiosis-specific effector kinase Mek1. This work shows that constitutive activation of Rad51 in Phase 1 results in a subset of DSBs being repaired by a Rad51-mediated interhomolog recombination pathway that is distinct from that of Dmc1. Strand invasion intermediates generated by Rad51 require more time to be processed into recombinants, resulting in a meiotic recombination checkpoint delay in prophase I. Without the checkpoint, Rad51-generated intermediates are more likely to involve a sister chromatid, thereby increasing Meiosis I chromosome nondisjunction. This Rad51 interhomolog recombination pathway is specifically promoted by the conserved 5'-3' helicase PIF1 and its paralog, RRM3 and requires Pif1 helicase activity and its interaction with PCNA. This work demonstrates that (1) inhibition of Rad51 during Phase 1 is important to prevent competition with Dmc1 for DSB repair, (2) Rad51-mediated meiotic recombination intermediates are initially processed differently than those made by Dmc1, and (3) the meiotic recombination checkpoint provides time during prophase 1 for processing of Rad51-generated recombination intermediates. Author summary: To sexually reproduce, cells containing two copies of each chromosome must undergo the specialized cell division of meiosis to sort the chromosomes into gametes containing a single copy of each chromosome. This reduction in chromosome number occurs by having one round of chromosome duplication followed by two rounds of chromosome segregation. At the first meiotic division, homologs segregate to opposite poles of the spindle. Homologous chromosomes identify each other through recombination which, along with sister chromatid cohesion, also serves to physically connect homologs to promote their proper segregation at Meiosis I. Interhomolog recombination is a process in which double strand breaks on one chromosome are converted to single stranded ends that can search for the complementary sequence on the homolog. In yeast and mammals, this homology search involves binding of single strand ends by two highly conserved recombinases, Rad51 and the meiosis specific Dmc1. Rad51 is used in mitotic cells to repair breaks, primarily using sister chromatids as templates, while Dmc1 functions in meiosis to generate interhomolog crossovers. In budding yeast, Rad51 strand exchange activity is normally inhibited while Dmc1 is active. We show here that when Rad51 and Dmc1 are active at the same time, Rad51 competes with Dmc1 to mediate interhomolog recombination of a subset of double strand breaks. However, because Rad51-generated recombination intermediates take longer to process, there is a need to keep Rad51 inactive while interhomolog recombination is occurring. [ABSTRACT FROM AUTHOR]

DNA replication is a vulnerable time for genome stability maintenance. Intrinsic stressors, as well as oncogenic stress, can challenge replication by fostering conflicts with transcription and stabilizing DNA:RNA hybrids. RAD18 is an E3 ubiquitin ligase for PCNA that is involved in coordinating DNA damage tolerance pathways to preserve genome stability during replication. In this study, we show that RAD18 deficient cells have higher levels of transcription-replication conflicts and accumulate DNA:RNA hybrids that induce DNA double strand breaks and replication stress. We find that these effects are driven in part by failure to recruit the Fanconi Anemia protein FANCD2 at difficult to replicate and R-loop prone genomic sites. FANCD2 activation caused by splicing inhibition or aphidicolin treatment is critically dependent on RAD18 activity. Thus, we highlight a RAD18-dependent pathway promoting FANCD2-mediated suppression of R-loops and transcription-replication conflicts. Author summary: Genome instability, a state in which cells acquire mutations more quickly, drives cancer initiation and progression. DNA is normally protected from such mutations by a host of specialized factors that recognize and repair potentially damaging circumstances. Here we describe under-appreciated links between a protein called RAD18, which recognizes potentially damaging stress, and a phenomenon called a transcription-replication conflict. If not repaired such conflicts can lead to DNA breaks. We find that RAD18 plays an important role in recruiting another repair protein called FANCD2 to sites of transcription-replication conflicts. By doing so, RAD18 protects the human genome from damage when it is faced with stresses such as those seen in cancer cells. This work provides new insight to how the FANCD2 protein is brought to potentially damaging sites in our genomes. [ABSTRACT FROM AUTHOR]

The germline mobilization of transposable elements (TEs) by small RNA mediated silencing pathways is conserved across eukaryotes and critical for ensuring the integrity of gamete genomes. However, genomes are recurrently invaded by novel TEs through horizontal transfer. These invading TEs are not targeted by host small RNAs, and their unregulated activity can cause DNA damage in germline cells and ultimately lead to sterility. Here we use hybrid dysgenesis—a sterility syndrome of Drosophila caused by transposition of invading P-element DNA transposons—to uncover host genetic variants that modulate dysgenic sterility. Using a panel of highly recombinant inbred lines of Drosophila melanogaster, we identified two linked quantitative trait loci (QTL) that determine the severity of dysgenic sterility in young and old females, respectively. We show that ovaries of fertile genotypes exhibit increased expression of splicing factors that suppress the production of transposase encoding transcripts, which likely reduces the transposition rate and associated DNA damage. We also show that fertile alleles are associated with decreased sensitivity to double-stranded breaks and enhanced DNA repair, explaining their ability to withstand high germline transposition rates. Together, our work reveals a diversity of mechanisms whereby host genotype modulates the cost of an invading TE, and points to genetic variants that were likely beneficial during the P-element invasion. Author summary: Transposable elements (TEs) are mobile genetic parasites that spread through host species' genomes by making additional copies of themselves in developing gametes. Transposition requires the breakage of DNA to introduce a new TE copy, which burdens the host cell to repair the damage or tolerate the mutation. To avoid these fitness costs, eukaryotic hosts suppress transposition in developing gametes through small-RNA mediated silencing. However, what happens when a new TE that is not recognized by existing small-RNAs invades the genome? We examined genetic variation in the degree to which Drosophila melanogaster oogenesis is disrupted by the transposition of a newly invading TE family. We show that while transposition results in gamete loss in some genotypes, other genotypes maintain fertility. Furthermore, we show that increased fertility likely reflects both decreased permissivity of transposition, as well as increased tolerance of DNA damage. Our observations provide a window into how host genetic variation impacts the consequences of invading TEs. [ABSTRACT FROM AUTHOR]

Homologous recombination (HR) plays an essential role in the maintenance of genome stability by promoting the repair of cytotoxic DNA double strand breaks (DSBs). More recently, the HR pathway has emerged as a core component of the response to replication stress, in part by protecting stalled replication forks from nucleolytic degradation. In that regard, the mammalian RAD51 paralogs (RAD51B, RAD51C, RAD51D, XRCC2, and XRCC3) have been involved in both HR-mediated DNA repair and collapsed replication fork resolution. Still, it remains largely obscure how they participate in both processes, thereby maintaining genome stability and preventing cancer development. To gain better insight into their contribution in cellulo, we mapped the proximal interactome of the classical RAD51 paralogs using the BioID approach. Aside from identifying the well-established BCDX2 and CX3 sub-complexes, the spliceosome machinery emerged as an integral component of our proximal mapping, suggesting a crosstalk between this pathway and the RAD51 paralogs. Furthermore, we noticed that factors involved RNA metabolic pathways are significantly modulated within the BioID of the classical RAD51 paralogs upon exposure to hydroxyurea (HU), pointing towards a direct contribution of RNA processing during replication stress. Importantly, several members of these pathways have prognostic potential in breast cancer (BC), where their RNA expression correlates with poorer patient outcome. Collectively, this study uncovers novel functionally relevant partners of the different RAD51 paralogs in the maintenance of genome stability that could be used as biomarkers for the prognosis of BC. Author summary: DNA double-strand breaks (DSBs) are highly cytotoxic DNA lesions that can compromise genomic instability, thereby driving carcinogenesis. DNA repair by homologous recombination (HR) is critical in faithfully repairing this type of DNA damage and the RAD51 paralogs (RAD51B, RAD51C, RAD51D, XRCC2, and XRCC3) have emerged as critical regulators of this pathway. Still, it remains largely unclear how they promote HR-mediate DNA repair. Here, we mapped their respective proximal interactome using the BioID approach and we identified the spliceosome machinery as an integral component of our proximal mapping. Importantly, several members of the spliceosome machinery have prognostic potential in breast cancer (BC), where their RNA expression correlates with poorer patient outcome. [ABSTRACT FROM AUTHOR]

Female reproductive aging is associated with decreased oocyte quality and fertility. The nematode Caenorhabditis elegans is a powerful system for understanding the biology of aging and exhibits age-related reproductive defects that are analogous to those observed in many mammals, including dysregulation of DNA repair. C. elegans germline function is influenced simultaneously by both reproductive aging and signals triggered by limited supplies of sperm, which are depleted over chronological time. To delineate the causes of DNA repair defects in aged C. elegans germlines, we assessed both DNA double strand break (DSB) induction and repair during meiotic prophase I progression in aged germlines which were depleted of self-sperm, mated, or never exposed to sperm. We find that germline DSB induction is dramatically reduced only in hermaphrodites which have exhausted their endogenous sperm, suggesting that a signal due specifically to sperm depletion downregulates DSB formation. We also find that DSB repair is delayed in aged germlines regardless of whether hermaphrodites had either a reduction in sperm supply or an inability to endogenously produce sperm. These results demonstrate that in contrast to DSB induction, DSB repair defects are a feature of C. elegans reproductive aging independent of sperm presence. Finally, we demonstrate that the E2 ubiquitin-conjugating enzyme variant UEV-2 is required for efficient DSB repair specifically in young germlines, implicating UEV-2 in the regulation of DNA repair during reproductive aging. In summary, our study demonstrates that DNA repair defects are a feature of C. elegans reproductive aging and uncovers parallel mechanisms regulating efficient DSB formation in the germline. Author summary: Aging leads to a decline in the quality of the female reproductive cells, known as oocytes. Oocytes subjected to reproductive aging experience an increase in both infertility and aneuploidies that cause miscarriages and birth defects. The nematode Caenorhabditis elegans is a classic model system used to determine the mechanisms of aging. Old C. elegans oocytes accrue many defects which may contribute to their reduced quality, including dysregulation of DNA repair. C. elegans fertility and germline function is also regulated oocyte-independently by sperm-dependent signals. To determine how aging and sperm may independently impact DNA repair in aging C. elegans oocytes, we control oocyte aging and sperm presence independently to evaluate their effects on DNA break formation and repair. We find that running out of sperm reduces the levels of DNA breaks which are produced, but the efficiency of DNA repair declines during aging independent of sperm effects. We also identify a protein which specifically promotes DNA repair in the oocytes of young animals, suggesting that this protein may regulate DNA repair in the germline during aging. Taken together, our research defines aging-specific and aging-independent mechanisms which regulate the genome integrity of oocytes. [ABSTRACT FROM AUTHOR]

Chromatin insulators are responsible for orchestrating long-range interactions between enhancers and promoters throughout the genome and align with the boundaries of Topologically Associating Domains (TADs). Here, we demonstrate an association between gypsy insulator proteins and the phosphorylated histone variant H2Av (γH2Av), normally a marker of DNA double strand breaks. Gypsy insulator components colocalize with γH2Av throughout the genome, in polytene chromosomes and in diploid cells in which Chromatin IP data shows it is enriched at TAD boundaries. Mutation of insulator components su(Hw) and Cp190 results in a significant reduction in γH2Av levels in chromatin and phosphatase inhibition strengthens the association between insulator components and γH2Av and rescues γH2Av localization in insulator mutants. We also show that γH2Av, but not H2Av, is a component of insulator bodies, which are protein condensates that form during osmotic stress. Phosphatase activity is required for insulator body dissolution after stress recovery. Together, our results implicate the H2A variant with a novel mechanism of insulator function and boundary formation. Author summary: The DNA in eukaryotic genomes is folded into domains called Topologically Associating Domains (TADS), which promote gene specific transcription regulation. Insulator proteins are DNA binding proteins that bind at the boundaries between adjacent TADs. Loop extrusion is a mechanism by which insulators promote TAD formation in vertebrate genomes, but the mechanism by which insulator proteins facilitate the formation of boundaries in Drosophila is not well understood. In this wok we show that there is an association between Drosophila Gypsy insulator proteins and the phosphorylated version of the histone variant H2Av (γH2Av). γH2Av has been traditionally linked to the mechanism of DNA repair, but our data shows that Gypsy insulator components colocalize with γH2Av throughout the genome, and that γH2Av is also enriched at TAD boundaries. Mutation of genes encoding insulator proteins su(Hw) and Cp190 results in a significant reduction in γH2Av levels, and inhibition of the phosphatase activity that removes phosphate from γH2Av strengthens the association between insulator proteins and γH2Av. We also show that γH2Av is a component of insulator bodies, which are protein condensates that form during osmotic stress. Together, our results implicate the H2A variant with a novel mechanism of insulator function and boundary formation. [ABSTRACT FROM AUTHOR]

Homologous recombination (HR) is a double-strand break DNA repair pathway that preserves chromosome structure. To repair damaged DNA, HR uses an intact donor DNA sequence located elsewhere in the genome. After the double-strand break is repaired, DNA sequence information can be transferred between donor and recipient DNA molecules through different mechanisms, including DNA crossovers that form between homologous chromosomes. Regulation of DNA sequence transfer is an important step in effectively completing HR and maintaining genome integrity. For example, mitotic exchange of information between homologous chromosomes can result in loss-of-heterozygosity (LOH), and in higher eukaryotes, the development of cancer. The DNA motor protein Rdh54 is a highly conserved DNA translocase that functions during HR. Several existing phenotypes in rdh54Δ strains suggest that Rdh54 may regulate effective exchange of DNA during HR. In our current study, we used a combination of biochemical and genetic techniques to dissect the role of Rdh54 on the exchange of genetic information during DNA repair. Our data indicate that RDH54 regulates DNA strand exchange by stabilizing Rad51 at an early HR intermediate called the displacement loop (D-loop). Rdh54 acts in opposition to Rad51 removal by the DNA motor protein Rad54. Furthermore, we find that expression of a catalytically inactivate allele of Rdh54, rdh54K318R, favors non-crossover outcomes. From these results, we propose a model for how Rdh54 may kinetically regulate strand exchange during homologous recombination. Author summary: Homologous recombination is an important pathway in repairing DNA double strand breaks. For the purposes of this study, HR can be divided into two stages. The first is a DNA repair stage in which the broken DNA molecule is fixed. In the second stage, information can move from one DNA molecule to another. Enzymes that use the power of ATP hydrolysis to move along dsDNA aid in regulating both stages of HR. In this work we focused on the understudied DNA motor protein Rdh54. We combined genetic and biochemical approaches to show that Rdh54 regulates HR by stabilizing the recombinase protein Rad51 at early HR intermediates. [ABSTRACT FROM AUTHOR]

Using budding yeast, we have studied Rad51-dependent break-induced replication (BIR), where the invading 3' end of a site-specific double-strand break (DSB) and a donor template share 108 bp of homology that can be easily altered. BIR still occurs about 10% as often when every 6th base is mismatched as with a perfectly matched donor. Here we explore the tolerance of mismatches in more detail, by examining donor templates that each carry 10 mismatches, each with different spatial arrangements. Although 2 of the 6 arrangements we tested were nearly as efficient as the evenly-spaced reference, 4 were significantly less efficient. A donor with all 10 mismatches clustered at the 3' invading end of the DSB was not impaired compared to arrangements where mismatches were clustered at the 5' end. Our data suggest that the efficiency of strand invasion is principally dictated by thermodynamic considerations, i.e., by the total number of base pairs that can be formed; but mismatch position-specific effects are also important. We also addressed an apparent difference between in vitro and in vivo strand exchange assays, where in vitro studies had suggested that at a single contiguous stretch of 8 consecutive bases was needed to be paired for stable strand pairing, while in vivo assays using 108-bp substrates found significant recombination even when every 6th base was mismatched. Now, using substrates of either 90 or 108 nt–the latter being the size of the in vivo templates–we find that in vitro D-loop results are very similar to the in vivo results. However, there are still notable differences between in vivo and in vitro assays that are especially evident with unevenly-distributed mismatches. Mismatches in the donor template are incorporated into the BIR product in a strongly polar fashion up to ~40 nucleotides from the 3' end. Mismatch incorporation depends on the 3'→ 5' proofreading exonuclease activity of DNA polymerase δ, with little contribution from Msh2/Mlh1 mismatch repair proteins, or from Rad1-Rad10 flap nuclease or the Mph1 helicase. Surprisingly, the probability of a mismatch 27 nt from the 3' end being replaced by donor sequence was the same whether the preceding 26 nucleotides were mismatched every 6th base or fully homologous. These data suggest that DNA polymerase δ "chews back" the 3' end of the invading strand without any mismatch-dependent cues from the strand invasion structure. However, there appears to be an alternative way to incorporate a mismatch at the first base at the 3' end of the donor. Author summary: DNA double-strand breaks (DSBs) are the most lethal forms of DNA damage and inaccurate repair of these breaks presents a serious threat to genomic integrity and cell viability. Break-induced replication (BIR) is a homologous recombination pathway that results in a nonreciprocal translocation of chromosome ends. We used budding yeast Saccharomyces cerevisiae to investigate Rad51-mediated BIR, where the invading 3' end of the DSB and a donor template share 108 bp of homology. We examined the tolerance of differently distributed mismatches on a homologous donor template. A donor with all 10 mismatches clustered every 6th base at the 3' invading end of the DSB was not impaired compared to arrangements where mismatches were clustered at the 5' end. We also compared the efficiency of in vivo BIR with in vitro D-loop formation and find that for substrates of the same length, the tolerance for mismatches is comparable. However, there are still notable differences between in vivo and in vitro assays that are especially evident in substrates with unevenly-distributed mismatches. Mismatches are incorporated into the BIR product in a strongly polar fashion as far as about 40 nucleotides from the 3' end, dependent on the 5' to 3' proofreading activity of DNA polymerase δ. Pol δ can "chew back" the 3' end of the invading strand even when the sequences removed have no mismatches for the first 26 nucleotides. However, a mismatch at the first base can be removed from the 3' end by another, unidentified mechanism. [ABSTRACT FROM AUTHOR]

Ensuring balanced distribution of chromosomes in gametes, meiotic recombination is essential for fertility in most sexually reproducing organisms. The repair of the programmed DNA double strand breaks that initiate meiotic recombination requires two DNA strand-exchange proteins, RAD51 and DMC1, to search for and invade an intact DNA molecule on the homologous chromosome. DMC1 is meiosis-specific, while RAD51 is essential for both mitotic and meiotic homologous recombination. DMC1 is the main catalytically active strand-exchange protein during meiosis, while this activity of RAD51 is downregulated. RAD51 is however an essential cofactor in meiosis, supporting the function of DMC1. This work presents a study of the mechanism(s) involved in this and our results point to DMC1 being, at least, a major actor in the meiotic suppression of the RAD51 strand-exchange activity in plants. Ectopic expression of DMC1 in somatic cells renders plants hypersensitive to DNA damage and specifically impairs RAD51-dependent homologous recombination. DNA damage-induced RAD51 focus formation in somatic cells is not however suppressed by ectopic expression of DMC1. Interestingly, DMC1 also forms damage-induced foci in these cells and we further show that the ability of DMC1 to prevent RAD51-mediated recombination is associated with local assembly of DMC1 at DNA breaks. In support of our hypothesis, expression of a dominant negative DMC1 protein in meiosis impairs RAD51-mediated DSB repair. We propose that DMC1 acts to prevent RAD51-mediated recombination in Arabidopsis and that this down-regulation requires local assembly of DMC1 nucleofilaments. Author summary: Essential for fertility and responsible for a major part of genetic variation in sexually reproducing species, meiotic recombination establishes the physical linkages between homologous chromosomes which ensure their balanced segregation in the production of gametes. These linkages, or chiasmata, result from DNA strand exchange catalyzed by the RAD51 and DMC1 recombinases and their numbers and distribution are tightly regulated. Essential for maintaining chromosomal integrity in mitotic cells, the strand-exchange activity of RAD51 is downregulated in meiosis, where it plays a supporting role to the activity of DMC1. Notwithstanding considerable attention from the genetics community, precisely why this is done and the mechanisms involved are far from being fully understood. We show here in the plant Arabidopsis that DMC1 can downregulate RAD51 strand-exchange activity and propose that this may be a general mechanism for suppression of RAD51-mediated recombination in meiosis. [ABSTRACT FROM AUTHOR]

During meiosis, DNA double-strand breaks (DSBs) occur throughout the genome, a subset of which are repaired to form reciprocal crossovers between chromosomes. Crossovers are essential to ensure balanced chromosome segregation and to create new combinations of genetic variation. Meiotic DSBs are formed by a topoisomerase-VI-like complex, containing catalytic (e.g. SPO11) proteins and auxiliary (e.g. PRD3) proteins. Meiotic DSBs are formed in chromatin loops tethered to a linear chromosome axis, but the interrelationship between DSB-promoting factors and the axis is not fully understood. Here, we study the localisation of SPO11-1 and PRD3 during meiosis, and investigate their respective functions in relation to the chromosome axis. Using immunocytogenetics, we observed that the localisation of SPO11-1 overlaps relatively weakly with the chromosome axis and RAD51, a marker of meiotic DSBs, and that SPO11-1 recruitment to chromatin is genetically independent of the axis. In contrast, PRD3 localisation correlates more strongly with RAD51 and the chromosome axis. This indicates that PRD3 likely forms a functional link between SPO11-1 and the chromosome axis to promote meiotic DSB formation. We also uncovered a new function of SPO11-1 in the nucleation of the synaptonemal complex protein ZYP1. We demonstrate that chromosome co-alignment associated with ZYP1 deposition can occur in the absence of DSBs, and is dependent on SPO11-1, but not PRD3. Lastly, we show that the progression of meiosis is influenced by the presence of aberrant chromosomal connections, but not by the absence of DSBs or synapsis. Altogether, our study provides mechanistic insights into the control of meiotic DSB formation and reveals diverse functional interactions between SPO11-1, PRD3 and the chromosome axis. Author summary: Most eukaryotes rely on the formation of gametes with half the number of chromosomes for sexual reproduction. Meiosis is a specialised type of cell division essential for the transition between a diploid and a haploid stage during gametogenesis. In early meiosis, programmed-DNA double strand breaks (DSBs) occur across the genome. These DSBs are processed by a set of proteins and the broken ends are repaired using the genetic information from the homologous chromosomes. These reciprocal exchanges of information between two chromosomes are called crossovers. Crossovers physical link chromosomes in pair which is essential to ensure their correct segregation during the two rounds of meiotic division. Crossovers are also essential for the creation of genetic diversity as they break genetic linkages to form novel allelic blocks. The formation of DSBs is not completely understood in plants. Here we studied the function of SPO11-1 and PRD3, two proteins involved in the formation of DSBs in Arabidopsis. We discovered functional differences in their respective mode of recruitment to the chromosomes, their interactions with proteins forming the chromosome core and their roles in chromosome co-alignment. These indicate that, although the SPO11-1 and PRD3 share a role in the formation of DSBs, the two proteins have additional and distinct roles beside DSB formation. [ABSTRACT FROM AUTHOR]

Recombinases RAD51 and its meiosis-specific paralog DMC1 accumulate on single-stranded DNA (ssDNA) of programmed DNA double strand breaks (DSBs) in meiosis. Here we used three-color dSTORM microscopy, and a mouse model with severe defects in meiotic DSB formation and synapsis (Hormad1-/-) to obtain more insight in the recombinase accumulation patterns in relation to repair progression. First, we used the known reduction in meiotic DSB frequency in Hormad1-/- spermatocytes to be able to conclude that the RAD51/DMC1 nanofoci that preferentially localize at distances of ~300 nm form within a single DSB site, whereas a second preferred distance of ~900 nm, observed only in wild type, represents inter-DSB distance. Next, we asked whether the proposed role of HORMAD1 in repair inhibition affects the RAD51/DMC1 accumulation patterns. We observed that the two most frequent recombinase configurations (1 DMC1 and 1 RAD51 nanofocus (D1R1), and D2R1) display coupled frequency dynamics over time in wild type, but were constant in the Hormad1-/- model, indicating that the lifetime of these intermediates was altered. Recombinase nanofoci were also smaller in Hormad1-/- spermatocytes, consistent with changes in ssDNA length or protein accumulation. Furthermore, we established that upon synapsis, recombinase nanofoci localized closer to the synaptonemal complex (SYCP3), in both wild type and Hormad1-/- spermatocytes. Finally, the data also revealed a hitherto unknown function of HORMAD1 in inhibiting coil formation in the synaptonemal complex. SPO11 plays a similar but weaker role in coiling and SYCP1 had the opposite effect. Using this large super-resolution dataset, we propose models with the D1R1 configuration representing one DSB end containing recombinases, and the other end bound by other ssDNA binding proteins, or both ends loaded by the two recombinases, but in below-resolution proximity. This may then often evolve into D2R1, then D1R2, and finally back to D1R1, when DNA synthesis has commenced. Author summary: In order to correctly pair homologous chromosomes in the first meiotic prophase, repair of programmed double strand breaks (DSBs) is essential. By unravelling molecular details of the protein assemblies at single DSBs, using super-resolution microscopy, we aim to understand the dynamics of repair intermediates and their functions. We investigated the localization of the two recombinases RAD51 and DMC1 in wild type and HORMAD1-deficient cells. HORMAD1 is involved in multiple aspects of homologous chromosome association: it regulates formation and repair of DSBs, and it stimulates formation of the synaptonemal complex (SC), the macromolecular protein assembly that connects paired chromosomes. RAD51 and DMC1 enable chromosome pairing by promoting the invasions of the intact chromatids by single-stranded DNA ends that result from DSBs. We found that in absence of HORMAD1, RAD51 and DMC1 showed small but significant morphological and positional changes, combined with altered kinetics of specific RAD51/DMC1 configurations. We also determined that there is a generally preferred distance of ~900 nm between meiotic DSBs along the SC. Finally, we observed changes in the structure of the SC in Hormad1-/- spermatocytes. This study contributes to a better understanding of the molecular details of meiotic homologous recombination and the role of HORMAD1 in meiotic prophase. [ABSTRACT FROM AUTHOR]

Break-induced replication (BIR) is a highly mutagenic eukaryotic homologous DNA recombination pathway that repairs one-ended DNA double strand breaks such as broken DNA replication forks and eroded telomeres. While searching for cis-acting factors regulating ectopic BIR efficiency, we found that ectopic BIR efficiency is the highest close to chromosome ends. The variations of ectopic BIR efficiency as a function of the length of DNA to replicate can be described as a combination of two decreasing exponential functions, a property in line with repeated cycles of strand invasion, elongation and dissociation that characterize BIR. Interestingly, the apparent processivity of ectopic BIR depends on the length of DNA already synthesized. Ectopic BIR is more susceptible to disruption during the synthesis of the first ~35–40 kb of DNA than later, notably when the template chromatid is being transcribed or heterochromatic. Finally, we show that the Srs2 helicase promotes ectopic BIR from both telomere proximal and telomere distal regions in diploid cells but only from telomere proximal sites in haploid cells. Altogether, we bring new light on the factors impacting a last resort DNA repair pathway. Author summary: DNA is a long molecule composed of two anti-parallel strands that can undergo breaks that need to be efficiently repaired to ensure genomic stability, hence preventing genetic diseases such as cancer. Homologous recombination is a major DNA repair pathway that copies DNA from intact homologous templates to seal DNA double strand breaks. Short DNA repair tracts are favored when homologous sequences for the two extremities of the broken molecule are present. However, when homologous sequences are present for only one extremity of the broken molecule, DNA repair synthesis can proceed up to the end of the chromosome, the telomere. This notably occurs at eroded telomeres when telomerase, the enzyme normally responsible for telomere elongation, is inactive, and at broken DNA replication intermediates. However, this Break-Induced Replication or BIR pathway is highly mutagenic. By initiating BIR at various distances from the telomere, we found that the length of DNA to synthesize significantly reduces BIR efficiency. Interestingly, our findings support two DNA synthesis phases, the first one being much less processive than the second one. Ultimately, this tends to restrain the use of this last resort DNA repair pathway to chromosome extremities notably when it takes place between non-allelic homologous sequences. [ABSTRACT FROM AUTHOR]

The NuA4 histone acetyltransferase complex, apart from its known role in gene regulation, has also been directly implicated in the repair of DNA double-strand breaks (DSBs), favoring homologous recombination (HR) in S/G2 during the cell cycle. Here, we investigate the antagonistic relationship of NuA4 with non-homologous end joining (NHEJ) factors. We show that budding yeast Rad9, the 53BP1 ortholog, can inhibit NuA4 acetyltransferase activity when bound to chromatin in vitro. While we previously reported that NuA4 is recruited at DSBs during the S/G2 phase, we can also detect its recruitment in G1 when genes for Rad9 and NHEJ factors Yku80 and Nej1 are mutated. This is accompanied with the binding of single-strand DNA binding protein RPA and Rad52, indicating DNA end resection in G1 as well as recruitment of the HR machinery. This NuA4 recruitment to DSBs in G1 depends on Mre11-Rad50-Xrs2 (MRX) and Lcd1/Ddc2 and is linked to the hyper-resection phenotype of NHEJ mutants. It also implicates NuA4 in the resection-based single-strand annealing (SSA) repair pathway along Rad52. Interestingly, we identified two novel non-histone acetylation targets of NuA4, Nej1 and Yku80. Acetyl-mimicking mutant of Nej1 inhibits repair of DNA breaks by NHEJ, decreases its interaction with other core NHEJ factors such as Yku80 and Lif1 and favors end resection. Altogether, these results establish a strong reciprocal antagonistic regulatory function of NuA4 and NHEJ factors in repair pathway choice and suggests a role of NuA4 in alternative repair mechanisms in situations where some DNA-end resection can occur in G1. Author summary: DNA double-strand breaks (DSBs) are one of the most harmful form of DNA damage. Cells employ two major repair pathways to resolve DSBs: Homologous Recombination (HR) and Non-Homologous End Joining (NHEJ). Here we wanted to dissect further the role played by the NuA4 (Nucleosome acetyltransferase of histone H4) complex in the repair of DSBs. Budding yeast NuA4 complex, like its mammalian homolog TIP60 complex, has been shown to favor repair by HR. Here, we show that indeed budding yeast NuA4 and components of the NHEJ repair pathway share an antagonistic relationship. Deletion of NHEJ components enables increased recruitment of NuA4 in the vicinity of DSBs, possible through two independent mechanisms, where NuA4 favors the end resection process which implicates it in repair by single-strand annealing (SSA), an alternate homology-based repair pathway. Additionally, we also present two NHEJ core components as new targets of NuA4 acetyltransferase activity and suggest that these acetylation events can disassemble the NHEJ repair complex from DSBs, favoring repair by HR. Our study demonstrates the importance of NuA4 in the modulation of DSB repair pathway choice. [ABSTRACT FROM AUTHOR]

Repair of DNA double-strand breaks (DSBs) by homologous recombination (HR) requires that the 5'-terminated DNA strands are resected to generate single-stranded DNA overhangs. This process is initiated by a short-range resection catalyzed by the MRX (Mre11-Rad50-Xrs2) complex, which is followed by a long-range step involving the nuclease Exo1 or Dna2. Here we show that the Saccharomyces cerevisiae ATP-dependent chromatin-remodeling protein Chd1 participates in both short- and long-range resection by promoting MRX and Exo1 association with the DSB ends. Furthermore, Chd1 reduces histone occupancy near the DSB ends and promotes DSB repair by HR. All these functions require Chd1 ATPase activity, supporting a role for Chd1 in the opening of chromatin at the DSB site to facilitate MRX and Exo1 processing activities. Author summary: DNA double strand breaks (DSBs) are among the most severe types of damage occurring in the genome because their faulty repair can result in chromosome instability, commonly associated with carcinogenesis. Efficient and accurate repair of DSBs relies on several proteins required to process them. However, eukaryotic genomes are compacted into chromatin, which restricts the access to DNA of the enzymes devoted to repair DNA DSBs. To overcome this natural barrier, eukaryotes have evolved chromatin remodeling enzymes that use energy derived from ATP hydrolysis to modulate chromatin structure. Here, we examine the role in DSB repair of the ATP-dependent chromatin remodeler Chd1, which is frequently mutated in prostate cancer. We find that Chd1 is important to repair DNA DSBs by homologous recombination (HR) because it promotes the association with a damaged site of the MRX complex and Exo1, which are necessary to initiate HR. This Chd1 function requires its ATPase activity, suggesting that Chd1 increases the accessibility to chromatin to initiate repair of DNA lesions. [ABSTRACT FROM AUTHOR]

The structural maintenance of chromosome (SMC) complex cohesin mediates sister chromatid cohesion established during replication, and damage-induced cohesion formed in response to DSBs post-replication. The translesion synthesis polymerase Polη is required for damage-induced cohesion through a hitherto unknown mechanism. Since Polη is functionally associated with transcription, and transcription triggers de novo cohesion in Schizosaccharomyces pombe, we hypothesized that transcription facilitates damage-induced cohesion in Saccharomyces cerevisiae. Here, we show dysregulated transcriptional profiles in the Polη null mutant (rad30Δ), where genes involved in chromatin assembly and positive transcription regulation were downregulated. In addition, chromatin association of RNA polymerase II was reduced at promoters and coding regions in rad30Δ compared to WT cells, while occupancy of the H2A.Z variant (Htz1) at promoters was increased in rad30Δ cells. Perturbing histone exchange at promoters inactivated damage-induced cohesion, similarly to deletion of the RAD30 gene. Conversely, altering regulation of transcription elongation suppressed the deficient damage-induced cohesion in rad30Δ cells. Furthermore, transcription inhibition negatively affected formation of damage-induced cohesion. These results indicate that the transcriptional deregulation of the Polη null mutant is connected with its reduced capacity to establish damage-induced cohesion. This also suggests a linkage between regulation of transcription and formation of damage-induced cohesion after replication. Author summary: The cohesin complex dynamically associates with chromosomes and holds sister chromatids together through cohesion established during replication. This ensures faithful chromosome segregation at anaphase. In budding yeast, DNA double strand breaks also trigger sister chromatid cohesion after replication. This so-called damage-induced cohesion is formed both close to the breaks, and genome-wide on undamaged chromosomes. The translesion synthesis polymerase eta (Polη) is specifically required for genome wide damage-induced cohesion. Although Polη is well characterized for its function in bypassing ultraviolet-induced DNA lesions, its mechanistic role in damage-induced cohesion is unclear. Here, we show that transcriptional regulation is perturbed in the persistent absence of Polη. We find that Polη preferably associates with certain types of promoters, although its role in transcription might be indirect. By testing mutants that perturb histone exchange or regulation of transcription, as well as through inhibiting transcription, we show that transcriptional deregulation negatively affects formation of damage-induced cohesion. This supports the connection between transcriptional deregulation and deficient damage-induced cohesion in the Polη null mutant. Importantly, our study provides new insight into formation of damage-induced cohesion after replication, which will be interesting to explore further. [ABSTRACT FROM AUTHOR]

Accurate repair of DNA double-strand breaks (DSBs) is crucial for cell survival and genome integrity. In Escherichia coli, DSBs are repaired by homologous recombination (HR), using an undamaged sister chromosome as template. The DNA intermediates of this pathway are expected to be branched molecules that may include 4-way structures termed Holliday junctions (HJs), and 3-way structures such as D-loops and repair forks. Using a tool creating a site-specific, repairable DSB on only one of a pair of replicating sister chromosomes, we have determined how these branched DNA intermediates are distributed across a DNA region that is undergoing DSB repair. In cells, where branch migration and cleavage of HJs are limited by inactivation of the RuvABC complex, HJs and repair forks are principally accumulated within a distance of 12 kb from sites of recombination initiation, known as Chi, on each side of the engineered DSB. These branched DNA structures can even be detected in the region of DNA between the Chi sites flanking the DSB, a DNA segment not expected to be engaged in recombination initiation, and potentially degraded by RecBCD nuclease action. This is observed even in the absence of the branch migration and helicase activities of RuvAB, RadA, RecG, RecQ and PriA. The detection of full-length DNA fragments containing HJs in this central region implies that DSB repair can restore the two intact chromosomes, into which HJs can relocate prior to their resolution. The distribution of recombination intermediates across the 12kb region beyond Chi is altered in xonA, recJ and recQ mutants suggesting that, in the RecBCD pathway of DSB repair, exonuclease I stimulates the formation of repair forks and that RecJQ promotes strand-invasion at a distance from the recombination initiation sites. Author summary: DNA double-strand breaks need to be accurately repaired to ensure cell survival. In the bacterium Escherichia coli, these breaks are repaired by the RecBCD pathway of homologous recombination, which involves copying genetic information from another intact and identical DNA template. During this repair process, the broken DNA and the template DNA form 4-way joint DNA molecules called Holliday junctions, and information lost from the broken chromosome is restored by DNA replication at three-way repair forks. Here, we have investigated the distribution of Holliday junctions and repair forks relative to a specific DNA double-strand break site in the E. coli chromosome, in cells where the movement and resolution of joint molecules is limited. Investigation of the impacts of Exonuclease I and the exonuclease-helicase RecJQ on the distribution of these joint molecules has led us to propose that, in the RecBCD pathway of homologous recombination, RecJQ can extend the region of single-stranded DNA on the broken chromosome and Exonuclease I can facilitate the formation of a repair fork by digesting the single-stranded DNA after it has paired with its template. [ABSTRACT FROM AUTHOR]

Homologous recombination is a high-fidelity repair pathway for DNA double-strand breaks employed during both mitotic and meiotic cell divisions. Such repair can lead to genetic exchange, originating from crossover (CO) generation. In mitosis, COs are suppressed to prevent sister chromatid exchange. Here, the BTR complex, consisting of the Bloom helicase (HIM-6 in worms), topoisomerase 3 (TOP-3), and the RMI1 (RMH-1 and RMH-2) and RMI2 scaffolding proteins, is essential for dismantling joint DNA molecules to form non-crossovers (NCOs) via decatenation. In contrast, in meiosis COs are essential for accurate chromosome segregation and the BTR complex plays distinct roles in CO and NCO generation at different steps in meiotic recombination. RMI2 stabilizes the RMI1 scaffolding protein, and lack of RMI2 in mitosis leads to elevated sister chromatid exchange, as observed upon RMI1 knockdown. However, much less is known about the involvement of RMI2 in meiotic recombination. So far, RMI2 homologs have been found in vertebrates and plants, but not in lower organisms such as Drosophila, yeast, or worms. We report the identification of the Caenorhabditis elegans functional homolog of RMI2, which we named RMIF-2. The protein shows a dynamic localization pattern to recombination foci during meiotic prophase I and concentration into recombination foci is mutually dependent on other BTR complex proteins. Comparative analysis of the rmif-2 and rmh-1 phenotypes revealed numerous commonalities, including in regulating CO formation and directing COs toward chromosome arms. Surprisingly, the prevalence of heterologous recombination was several fold lower in the rmif-2 mutant, suggesting that RMIF-2 may be dispensable or less strictly required for some BTR complex-mediated activities during meiosis. Author summary: Bloom syndrome is caused by mutations in proteins of the BTR complex (consisting of the Bloom helicase, topoisomerase 3, and the RMI1 and RMI2 scaffolding proteins) and the clinical characteristics are growth deficiency, short stature, skin photosensitivity, and increased cancer predisposition. At the cellular level, characteristic features are the presence of increased sister chromatid exchange on chromosomes; unresolved DNA recombination intermediates that eventually cause genome instability; and erroneous DNA repair by heterologous recombination (recombination between non-identical sequences, extremely rare in wild type animals), which can trigger translocations and chromosomal rearrangements. Identification of the Caenorhabditis elegans ortholog of RMI2 (called RMIF-2) allowed us to compare heterologous recombination in the germline of mutants of various BTR complex proteins. The heterologous recombination rate was several fold lower in rmif-2 mutants than in mutants of rmh-1 and him-6 (worm homologs of RMI1 and the Bloom helicase, respectively). Nevertheless, many phenotypic features point at RMIF-2 working together with RMH-1. If these germline functions of RMI2/RMIF-2 are conserved in humans, this might mean that individuals with RMI2 mutations have a lower risk of translocations and genome rearrangements than those with mutations in the other BTR complex genes. [ABSTRACT FROM AUTHOR]

Chromatin modifying complexes play important yet not fully defined roles in DNA repair processes. The essential NuA4 histone acetyltransferase (HAT) complex is recruited to double-strand break (DSB) sites and spreads along with DNA end resection. As predicted, NuA4 acetylates surrounding nucleosomes upon DSB induction and defects in its activity correlate with altered DNA end resection and Rad51 recombinase recruitment. Importantly, we show that NuA4 is also recruited to the donor sequence during recombination along with increased H4 acetylation, indicating a direct role during strand invasion/D-loop formation after resection. We found that NuA4 cooperates locally with another HAT, the SAGA complex, during DSB repair as their combined action is essential for DNA end resection to occur. This cooperation of NuA4 and SAGA is required for recruitment of ATP-dependent chromatin remodelers, targeted acetylation of repair factors and homologous recombination. Our work reveals a multifaceted and conserved cooperation mechanism between acetyltransferase complexes to allow repair of DNA breaks by homologous recombination. Author summary: DNA double strand breaks (DSBs) are among the most dangerous types of DNA lesions as they can produce genomic instability that leads to cancer and genetic diseases. It is therefor crucial to understand the precise molecular mechanisms used by cells to detect and repair this type of damages. Homologous recombination using sister chromatid as template is the most accurate pathway to repair these breaks but has to occur within the context of the DNA compacted structure in chromosomes. Here, we show that two enzymes, NuA4 and SAGA, that acetylate the structural components of chromosomes in the vicinity of the DNA breaks are together essential for recombination-mediated repair to occur. We found that they are recruited at an early step after damage detection and their action allows subsequent remodeling of local structural organisation by other enzymes, providing DNA access to the recombination machinery. These results highlight the cooperation of enzymes for a same goal, providing robustness in the repair process as only the loss of both leads to major defects. [ABSTRACT FROM AUTHOR]

DNA double-strand breaks arise in vivo when a dicentric chromosome (two centromeres on one chromosome) goes through mitosis with the two centromeres attached to opposite spindle pole bodies. Repair of the DSBs generates phenotypic diversity due to the range of monocentric derivative chromosomes that arise. To explore whether DSBs may be differentially repaired as a function of their spatial position in the chromosome, we have examined the structure of monocentric derivative chromosomes from cells containing a suite of dicentric chromosomes in which the distance between the two centromeres ranges from 6.5 kb to 57.7 kb. Two major classes of repair products, homology-based (homologous recombination (HR) and single-strand annealing (SSA)) and end-joining (non-homologous (NHEJ) and micro-homology mediated (MMEJ)) were identified. The distribution of repair products varies as a function of distance between the two centromeres. Genetic dependencies on double strand break repair (Rad52), DNA ligase (Lif1), and S phase checkpoint (Mrc1) are indicative of distinct repair pathway choices for DNA breaks in the pericentromeric chromatin versus the arms. Author summary: A challenge in chromosome biology is to integrate the linear code with spatial organization and chromosome dynamics within the nucleus. The major sub-division of function in the nucleus is the nucleolus, the site of ribosomal RNA synthesis. We report that the pericentromere DNA surrounding the centromere is another region of confined biochemistry. We have found that chromosome breaks between two centromeres that both lie within the pericentromeric region of the chromosomes are repaired via pathways that do not rely on sequence homology (MMEJ or NHEJ). Chromosome breaks in dicentric chromosomes whose centromeres are separated by > 20 kb are repaired via pathways that rely mainly on sequence homology (HR, SSA). The repair of breaks in the pericentromere versus breaks in the arms are differentially dependent on Rad52, Lif1, and Mrc1, further indicative of spatial control over DNA repair pathways. [ABSTRACT FROM AUTHOR]

Endogenous genotoxic stress occurs in healthy cells due to competition between DNA replication machinery, and transcription and topographic relaxation processes. This causes replication fork stalling and regression, which can further collapse to form single-ended double strand breaks (seDSBs). Super-resolution microscopy has made it possible to directly observe replication stress and DNA damage inside cells, however new approaches to sample preparation and analysis are required. Here we develop and apply multicolor single molecule microscopy to visualize individual replication forks under mild stress from the trapping of Topoisomerase I cleavage complexes, a damage induction which closely mimics endogenous replicative stress. We observe RAD51 and RAD52, alongside RECQ1, as the first responder proteins to stalled but unbroken forks, whereas Ku and MRE11 are initially recruited to seDSBs. By implementing novel super-resolution imaging assays, we are thus able to discern closely related replication fork stress motifs and their repair pathways. Author summary: Damage to the genetic code embedded in an organism's DNA can result in mutation or cell death which, in turn, can lead to disease and dysfunction. DNA damage is the main cause of many human diseases including cancer, and some forms of neurodegeneration and immune dysfunction. DNA double strand breaks (DSBs), which occur a handful of times in each replicating cell each day, are especially deleterious due to the difficulty of their repair. The main endogenous cause of DSBs is the breakdown of DNA replication forks however there is increasing evidence that damage and stress at these forks can also result in unbroken intermediate structures which avoid DSB formation such as fork regression. We have developed and applied new assays for labelling and visualizing damaged replication forks using super-resolution microscopy. This has enabled us to differentiate between broken and unbroken forks and to discern the different proteins that are recruited for DSB and regressed fork repair. Our data further demonstrate that these assays are widely applicable to DNA damage research and offer a new approach to mapping the spatiotemporal repair of individual damage events inside cells. [ABSTRACT FROM AUTHOR]

TDP-43 is a DNA and RNA binding protein involved in RNA processing and with structural resemblance to heterogeneous ribonucleoproteins (hnRNPs), whose depletion sensitizes neurons to double strand DNA breaks (DSBs). Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disorder, in which 97% of patients are familial and sporadic cases associated with TDP-43 proteinopathies and conditions clearing TDP-43 from the nucleus, but we know little about the molecular basis of the disease. After showing with the non-neuronal model of HeLa cells that TDP-43 depletion increases R loops and associated genome instability, we prove that mislocalization of mutated TDP-43 (A382T) in transfected neuronal SH-SY5Y and lymphoblastoid cell lines (LCLs) from an ALS patient cause R-loop accumulation, and R loop-dependent increased DSBs and Fanconi Anemia repair centers. These results uncover a new role of TDP-43 in the control of co-transcriptional R-loops and the maintenance of genome integrity by preventing harmful R-loop accumulation. Our findings thus link TDP-43 pathology to increased R-loops and R loop-mediated DNA damage opening the possibility that R-loop modulation in TDP-43-defective cells might help develop ALS therapies. Authors' summary: Amyotrophic Lateral Sclerosis (ALS) is an adult onset, progressive neurodegenerative disease, caused by the selective loss of upper and lower motor neurons in the cerebral cortex, brainstem and spinal cord. The nuclear TDP-43 RNA binding protein, is encoded by a major gene for ALS susceptibility whose mutations are found in 3% of familial and 2% of sporadic ALS cases. Thanks to its ability to recognize DNA and RNA, TDP-43 is involved in different steps of mRNA metabolism and in several mechanisms of genome integrity. This, together with the fact that R loops or DNA-RNA hybrids are a common source of genome instability, prompted us to investigate whether TDP-43 deficiency has any role in R loop homeostasis that could explain previously described DNA damage response defects of ALS cells. We show that TDP-43 plays a role in preventing R loop accumulation and associated genome instability in neuronal and non-neuronal cells, as well as in patient cell lines. Thus, our study opens the possibility that R-loop modulation in TDP-43-defective cells might help develop ALS therapies. [ABSTRACT FROM AUTHOR]

The Rad51/RecA family of recombinases perform a critical function in typical repair of double-strand breaks (DSBs): strand invasion of a resected DSB end into a homologous double-stranded DNA (dsDNA) template sequence to initiate repair. However, repair of a DSB using single stranded DNA (ssDNA) as a template, a common method of CRISPR/Cas9-mediated gene editing, is Rad51-independent. We have analyzed the genetic requirements for these Rad51-independent events in Saccharomyces cerevisiae by creating a DSB with the site-specific HO endonuclease and repairing the DSB with 80-nt single-stranded oligonucleotides (ssODNs), and confirmed these results by a Cas9-mediated DSBs in combination with a bacterial retron system that produces ssDNA templates in vivo. We show that single strand template repair (SSTR), is dependent on Rad52, Rad59, Srs2 and the Mre11-Rad50-Xrs2 (MRX) complex, but unlike other Rad51-independent recombination events, independent of Rdh54. We show that Rad59 acts to alleviate the inhibition of Rad51 on Rad52's strand annealing activity both in SSTR and in single strand annealing (SSA). Gene editing is Rad51-dependent when double-stranded oligonucleotides of the same size and sequence are introduced as templates. The assimilation of mismatches during gene editing is dependent on the activity of Msh2, which acts very differently on the 3' side of the ssODN which can anneal directly to the resected DSB end compared to the 5' end. In addition DNA polymerase Polδ's 3' to 5' proofreading activity frequently excises a mismatch very close to the 3' end of the template. We further report that SSTR is accompanied by as much as a 600-fold increase in mutations in regions adjacent to the sequences directly undergoing repair. These DNA polymerase ζ-dependent mutations may compromise the accuracy of gene editing. Author summary: DNA double strand breaks (DSBs) are one of the most lethal types of damage that can be inflicted on a chromosome and failure to repair such lesions can result in chromosome instability, commonly associated with human cancer. A knowledge of DNA repair mechanisms is also critical in the exploitation of gene therapy, a process that includes intentionally breaking the DNA to modify the genetic sequence. Here we compared two site-specific methods to create DSBs (HO endonuclease and CRISPR/Cas9) in budding yeast, to modify several DNA targets by single-strand DNA template repair (SSTR). We show that gene editing uses a DSB repair pathway that is independent of the canonical repair protein Rad51 and distinct from previously studied Rad51-independent pathways in its requirements for several other known recombination proteins. We show both in gene editing and in single-strand annealing that Rad59 acts to suppress the modulation of Rad52's strand annealing activity by Rad51. We also determined how mismatches in the template are incorporated into the genome, and that this assimilation reflects different aspects of Msh2-mediated mismatch repair as well as Polδ-mediated proofreading. These insights provide insight into the mechanisms of DSB repair by this important gene-editing pathway. [ABSTRACT FROM AUTHOR]

During meiosis, diploid organisms reduce their chromosome number by half to generate haploid gametes. This process depends on the repair of double strand DNA breaks as crossover recombination events between homologous chromosomes, which hold homologs together to ensure their proper segregation to opposite spindle poles during the first meiotic division. Although most organisms are limited in the number of crossovers between homologs by a phenomenon called crossover interference, the consequences of excess interfering crossovers on meiotic chromosome segregation are not well known. Here we show that extra interfering crossovers lead to a range of meiotic defects and we uncover mechanisms that counteract these errors. Using chromosomes that exhibit a high frequency of supernumerary crossovers in Caenorhabditis elegans, we find that essential chromosomal structures are mispatterned in the presence of multiple crossovers, subjecting chromosomes to improper spindle forces and leading to defects in metaphase alignment. Additionally, the chromosomes with extra interfering crossovers often exhibited segregation defects in anaphase I, with a high incidence of chromatin bridges that sometimes created a tether between the chromosome and the first polar body. However, these anaphase I bridges were often able to resolve in a LEM-3 nuclease dependent manner, and chromosome tethers that persisted were frequently resolved during Meiosis II by a second mechanism that preferentially segregates the tethered sister chromatid into the polar body. Altogether these findings demonstrate that excess interfering crossovers can severely impact chromosome patterning and segregation, highlighting the importance of limiting the number of recombination events between homologous chromosomes for the proper execution of meiosis. Author summary: Meiosis is a process that ensures developing eggs and sperm contain the correct number of chromosomes. Failure to accurately segregate chromosomes during meiosis is one of the leading causes of birth defects and miscarriages. During meiosis, crossover events must form between the homologous chromosomes to ensure proper chromosome segregation. Although crossover events are required for proper chromosome segregation in most organisms, crossover numbers are limited even when the meiotic cell is overloaded with DNA breaks, the initiating events for crossovers. This stringent limitation of crossovers in multiple organisms suggests that there are negative consequences to having too many crossovers, but this has not been formally tested. In this study, we find that increasing crossover number negatively impacts chromosome segregation during meiosis by altering chromosome-associated structures, which leads to misalignment of the chromosomes on the meiotic spindle. Moreover, chromosomes with excess crossovers often have large chromatin bridges during the chromosome segregation process, but we find that these bridges can be corrected by at least two mechanisms. Our results thus highlight the importance of limiting crossover numbers to enable faithful chromosome segregation during sperm and egg development. [ABSTRACT FROM AUTHOR]

In Paramecium tetraurelia, a large proportion of the germline genome is reproducibly removed from the somatic genome after sexual events via a process involving small (s)RNA-directed heterochromatin formation and DNA excision and repair. How germline limited DNA sequences are specifically recognized in the context of chromatin remains elusive. Here, we use a reverse genetics approach to identify factors involved in programmed genome rearrangements. We have identified a P. tetraurelia homolog of the highly conserved histone chaperone Spt16 subunit of the FACT complex, Spt16-1, and show its expression is developmentally regulated. A functional GFP-Spt16-1 fusion protein localized exclusively in the nuclei where genome rearrangements take place. Gene silencing of Spt16-1 showed it is required for the elimination of all germline-limited sequences, for the survival of sexual progeny, and for the accumulation of internal eliminated sequence (ies)RNAs, an sRNA population produced when elimination occurs. Normal accumulation of 25 nt scanRNAs and deposition of silent histone marks H3K9me3 and H3K27me3 indicated that Spt16-1 does not regulate the scanRNA-directed heterochromatin pathway involved in the early steps of DNA elimination. We further show that Spt16-1 is required for the correct nuclear localization of the PiggyMac (Pgm) endonuclease, which generates the DNA double-strand breaks required for DNA elimination. Thus, Spt16-1 is essential for Pgm function during programmed genome rearrangements. We propose a model in which Spt16-1 mediates interactions between the excision machinery and chromatin, facilitating endonuclease access to DNA cleavage sites during genome rearrangements. Author summary: The genome is generally similar in all the cells of an organism. However, in the ciliate Paramecium tetraurelia, massive and reproducible programmed DNA elimination leads to a highly streamlined somatic genome. In eukaryotes, DNA is packaged into nucleosomes, which ensure genome integrity but act as a barrier to enzymes acting on DNA. How the endonuclease PiggyMac gains access to the genome to initiate DNA elimination remains elusive. Here, we identified four P. tetraurelia genes encoding homologs of the conserved histone chaperone Spt16, which can modulate acesss to DNA by promoting nucleosome assembly and disassembly. We demonstrated that the most divergent gene, SPT16-1, has a highly specialized expression pattern, similar to that of PiggyMac, and a specific role in programmed DNA elimination. We show that the Spt16-1 protein, like PiggyMac, is exclusively localized in the differentiating somatic nucleus, and is also required for the dramatic elimination of germline-limited sequences. We further show that Spt16-1 directs the correct nuclear localization of the PiggyMac endonuclease. Thus, Spt16-1 is essential for PiggyMac function during programmed DNA elimination. We propose that Spt16-1 mediates the interaction between PiggyMac and chromatin or DNA, facilitating endonuclease access to DNA cleavage sites. [ABSTRACT FROM AUTHOR]

The recombinase RAD51, and its meiosis-specific paralog DMC1 localize at DNA double-strand break (DSB) sites in meiotic prophase. While both proteins are required during meiotic prophase, their spatial organization during meiotic DSB repair is not fully understood. Using super-resolution microscopy on mouse spermatocyte nuclei, we aimed to define their relative position at DSB foci, and how these vary in time. We show that a large fraction of meiotic DSB repair foci (38%) consisted of a single RAD51 nanofocus and a single DMC1 nanofocus (D1R1 configuration) that were partially overlapping with each other (average center-center distance around 70 nm). The vast majority of the rest of the foci had a similar large RAD51 and DMC1 nanofocus, but in combination with additional smaller nanofoci (D2R1, D1R2, D2R2, or DxRy configuration) at an average distance of around 250 nm. As prophase progressed, less D1R1 and more D2R1 foci were observed, where the large RAD51 nanofocus in the D2R1 foci elongated and gradually oriented towards the distant small DMC1 nanofocus. D1R2 foci frequency was relatively constant, and the single DMC1 nanofocus did not elongate, but was frequently observed between the two RAD51 nanofoci in early stages. D2R2 foci were rare (<10%) and nearest neighbour analyses also did not reveal cofoci formation between D1R1 foci. However, overall, foci localised nonrandomly along the SC, and the frequency of the distance distributions peaked at 800nm, indicating interference and/or a preferred distance between two ends of a DSB. DMC1 nanofoci where somewhat further away from the axial or lateral elements of the synaptonemal complex (SC, connecting the chromosomal axes of homologs) compared to RAD51 nanofoci. In the absence of the transverse filament of the SC, early configurations were more prominent, and RAD51 nanofocus elongation occurred only transiently. This in-depth analysis of single cell landscapes of RAD51 and DMC1 accumulation patterns at DSB repair sites at super-resolution revealed the variability of foci composition, and defined functional consensus configurations that change over time. Author summary: Meiosis is a specific type of cell division that is central to sperm and egg formation in sexual reproduction. It forms cells with a single copy of each chromosome, instead of the two copies that are normally present. In meiotic prophase I, homologous chromosomes must connect to each other, to be correctly distributed between the daughter cells. This involves the formation and repair of double-strand breaks in the DNA. Here we used super-resolution microscopy to elucidate the localization patterns of two important DNA repair proteins: RAD51 and DMC1. We found that repair sites most often contain a single large nanofocus of both proteins, with or without one additional smaller nanofocus of either protein. RAD51 protein nanofoci displayed lengthening as meiotic prophase progressed, and localised somewhat closer to the protein axis that mediates the physical connection (synapsis) between homologous chromosomes compared to DMC1 nanofoci. When chromosome synapsis was disturbed, we observed changes in the dynamics of protein accumulation patterns, indicating that they actually correspond to certain repair intermediates changing in relative frequency of occurrence. These analyses of single meiotic DNA repair foci reveal the biological variability in protein accumulation patterns, and the localization of RAD51 and DMC1 relative to each other, thereby contributing to our understanding of the molecular basis of meiotic homologous recombination. [ABSTRACT FROM AUTHOR]

During repair of DNA double-strand breaks, resection of DNA ends influences how these lesions will be repaired. If resection is activated, the break will be channeled through homologous recombination; if not, it will be simply ligated using the non-homologous end-joining machinery. Regulation of resection relies greatly on modulating CtIP, which can be done by modifying: i) its interaction partners, ii) its post-translational modifications, or iii) its cellular levels, by regulating transcription, splicing and/or protein stability/degradation. Here, we have analyzed the role of ALC1, a chromatin remodeler previously described as an integral part of the DNA damage response, in resection. Strikingly, we found that ALC1 affects resection independently of chromatin remodeling activity or its ability to bind damaged chromatin. In fact, it cooperates with the RNA-helicase eIF4A1 to help stabilize the most abundant splicing form of CtIP mRNA. This function relies on the presence of a specific RNA sequence in the 5′ UTR of CtIP. Therefore, we describe an additional layer of regulation of CtIP—at the level of mRNA stability through ALC1 and eIF4A1. Author summary: The DNA molecule is constantly threatened by the appearance of physical or chemical modifications that endanger the integrity of the genetic information. Among them, the breakage of the DNA molecule is the most challenging to deal with. In order to minimize such risks, cells have developed multiple DNA repair mechanisms that take care of broken chromosomes. The regulation of the usage of each different mechanism is extremely important for the safekeeping of the genetic material. CtIP is a key protein of pivotal importance in the decision between different broken DNA repair pathways. Here we show a novel regulatory mechanism that controls the abundance of this protein by controlling the stability of the different mRNA molecules produced by the CtIP gene. In brief, we show that the factors ALC1 and eIF4A1 affect the stability of some, but not all, CtIP mRNA variants. This effect is dependent on the presence or absence of a specific RNA structure, called a G-quartet or G-quadruplex, in a non-coding portion of the mRNA. [ABSTRACT FROM AUTHOR]

Bases within DNA are frequently damaged, producing obstacles to efficient and accurate DNA replication by replicative polymerases. Translesion synthesis (TLS) polymerases, via their ability to catalyze nucleotide additions to growing DNA chains across DNA lesions, promote replication of damaged DNA, thus preventing checkpoint activation, genome instability and cell death. In this study, we used C. elegans to determine the contribution of TLS activity on long-term stability of an animal genome. We monitored and compared the types of mutations that accumulate in REV1, REV3, POLH1 and POLK deficient animals that were grown under unchallenged conditions. We also addressed redundancies in TLS activity by combining all deficiencies. Remarkably, animals that are deficient for all Y-family polymerases as well as animals that have lost all TLS activity are viable and produce progeny, demonstrating that TLS is not essential for animal life. Whole genome sequencing analyses, however, reveal that TLS is needed to prevent genomic scars from accumulating. These scars, which are the product of polymerase theta-mediated end joining (TMEJ), are found overrepresented at guanine bases, consistent with TLS suppressing DNA double-strand breaks (DSBs) from occurring at replication-blocking guanine adducts. We found that in C. elegans, TLS across spontaneous damage is predominantly error free and anti-clastogenic, and thus ensures preservation of genetic information. Author summary: Research in the fields of DNA repair and mutagenesis has led to enormous insight into the mechanisms responsible for maintaining genetic integrity. However, which processes drive de novo mutations and will thus contribute to inherited diseases are still unclear. One process thought to underlie spontaneous mutagenesis is replication of damaged DNA by specialised so-called "Translesion synthesis" polymerases, which have the ability to replicate across damaged bases, but are not very accurate. To address the impact of TLS or the lack thereof on genome integrity, we have knocked out all TLS enzymes that are encoded by the C. elegans genome, individually and in combination, and monitored mutation accumulation during prolonged culturing of these animals without external sources of DNA damage. We found that TLS is not the major driver of spontaneous mutagenesis in this organism, however, it protects the genome from harmful small deletions that result from mutagenic repair of DNA breaks. We also found that, contrary to what was expected, TLS activity is not essential for reproduction in a multicellular organism with the tissue complexity and genome size of C. elegans. [ABSTRACT FROM AUTHOR]

Meiotic double-strand breaks (DSBs) are generated by the evolutionarily conserved SPO11 complex in the context of chromatin loops that are organized along axial elements (AEs) of chromosomes. However, how DSBs are formed with respect to chromosome axes and the SPO11 complex remains unclear in plants. Here, we confirm that DSB and bivalent formation are defective in maize spo11-1 mutants. Super-resolution microscopy demonstrates dynamic localization of SPO11-1 during recombination initiation, with variable numbers of SPO11-1 foci being distributed in nuclei but similar numbers of SPO11-1 foci being found on AEs. Notably, cytological analysis of spo11-1 meiocytes revealed an aberrant AE structure. At leptotene, AEs of wild-type and spo11-1 meiocytes were similarly curly and discontinuous. However, during early zygotene, wild-type AEs become uniform and exhibit shortened axes, whereas the elongated and curly AEs persisted in spo11-1 mutants, suggesting that loss of SPO11-1 compromised AE structural maturation. Our results reveal an interesting relationship between SPO11-1 loading onto AEs and the conformational remodeling of AEs during recombination initiation. Author summary: Meiosis is essential during sexual reproduction to produce haploid gametes. Recombination is the most crucial step during meiotic prophase I. It enables pairing of homologous chromosomes prior to their reductional division and generates new combinations of genetic alleles for transmission to the next generation. Meiotic recombination is initiated by generating DNA double-strand breaks (DSBs) via SPO11, a topoisomerase-related enzyme. The activity, timing and location of this DSB machinery must be controlled precisely, but how this is achieved remains obscure. Here, we show dynamic localization of SPO11-1 on chromatin during meiotic initiation in maize, yet a similar number of SPO11-1 is able to load onto axial elements (AEs), which accompanies a structural change of the AEs of wild-type meiotic chromosomes. Interestingly, loss of SPO11-1 not only affects DSB formation but also impairs structural alterations of AEs, resulting in abnormally long and curly AEs during early meiosis. Our study provides new insights into SPO11-1 localization during recombination initiation and suggests an intimate relationship between DSB formation and AE structural changes. [ABSTRACT FROM AUTHOR]

Gene duplication and diversification drive the emergence of novel functions during evolution. Because of whole genome duplications, ciliates from the Paramecium aurelia group constitute a remarkable system to study the evolutionary fate of duplicated genes. Paramecium species harbor two types of nuclei: a germline micronucleus (MIC) and a somatic macronucleus (MAC) that forms from the MIC at each sexual cycle. During MAC development, ~45,000 germline Internal Eliminated Sequences (IES) are excised precisely from the genome through a 'cut-and-close' mechanism. Here, we have studied the P. tetraurelia paralogs of KU80, which encode a key DNA double-strand break repair factor involved in non-homologous end joining. The three KU80 genes have different transcription patterns, KU80a and KU80b being constitutively expressed, while KU80c is specifically induced during MAC development. Immunofluorescence microscopy and high-throughput DNA sequencing revealed that Ku80c stably anchors the PiggyMac (Pgm) endonuclease in the developing MAC and is essential for IES excision genome-wide, providing a molecular explanation for the previously reported Ku-dependent licensing of DNA cleavage at IES ends. Expressing Ku80a under KU80c transcription signals failed to complement a depletion of endogenous Ku80c, indicating that the two paralogous proteins have distinct properties. Domain-swap experiments identified the α/β domain of Ku80c as the major determinant for its specialized function, while its C-terminal part is required for excision of only a small subset of IESs located in IES-dense regions. We conclude that Ku80c has acquired the ability to license Pgm-dependent DNA cleavage, securing precise DNA elimination during programmed rearrangements. The present study thus provides novel evidence for functional diversification of genes issued from a whole-genome duplication. Author summary: DNA double-strand breaks (DSBs) are highly hazardous lesions, which, if incorrectly repaired, may cause cancer or trigger cell death. In spite of their toxicity, programmed DSBs are part of important physiological processes, such as the mixing of paternal and maternal alleles during meiosis or the generation of antibody diversity during the immune response that follows an infection. Here, we have studied how DNA breakage by a specific endonuclease and DSB repair by the non-homologous end joining pathway are coordinated during the programmed elimination of several thousands of short DNA fragments, which takes place at each sexual cycle in the ciliate Paramecium tetraurelia. We show that the product of one specific copy among three duplicates of the DSB repair KU80 gene has the unique ability to tightly anchor the endonuclease in the nucleus and license programmed DNA cleavage at both ends of eliminated sequences. We identify the N-terminal domain of this Ku80 variant as the major determinant of its specific function. We propose that functional diversification of duplicated genes during evolution has allowed P. tetraurelia to secure precise programmed DNA elimination through the coupling of DSB introduction and repair. [ABSTRACT FROM AUTHOR]

FANCJ helicase mutations are known to cause hereditary breast and ovarian cancers as well as bone marrow failure syndrome Fanconi anemia. FANCJ plays an important role in the repair of DNA inter-strand crosslinks and DNA double-strand breaks (DSBs) by homologous recombination (HR). Nonetheless, the molecular mechanism by which FANCJ controls HR mediated DSB repair is obscure. Here, we show that FANCJ promotes DNA end resection by recruiting CtIP to the sites of DSBs. This recruitment of CtIP is dependent on FANCJ K1249 acetylation. Notably, FANCJ acetylation is dependent on FANCJ S990 phosphorylation by CDK. The CDK mediated phosphorylation of FANCJ independently facilitates its interaction with BRCA1 at damaged DNA sites and promotes DNA end resection by CtIP recruitment. Strikingly, mutational studies reveal that ATP binding competent but hydrolysis deficient FANCJ partially supports end resection, indicating that in addition to the scaffolding role of FANCJ in CtIP recruitment, its helicase activity is important for promoting end resection. Together, these data unravel a novel function of FANCJ helicase in DNA end resection and provide mechanistic insights into its role in repairing DSBs by HR and in genome maintenance. Author summary: Homologous recombination has been considered as an error-free pathway in repairing DSBs and maintaining genome stability. Cyclin-dependent kinases (CDKs) and various factors including MRE11, CtIP, EXO1, and BLM helicase participate in DNA end resection to promote HR mediated DSB repair. Despite the identification of FANCJ helicase role in HR and tumor suppression, the molecular mechanism by which FANCJ helicase participates in HR is obscure. Here, we show that FANCJ helicase controls DNA end resection by recruiting CtIP to the sites of DSBs. The loading of CtIP is dependent on FANCJ acetylation which is mediated by CDK dependent phosphorylation of FANCJ. Moreover, in addition to FANCJ mediated CtIP recruitment, its helicase activity is also essential for DNA end resection. Our data identify FANCJ as a novel player in the DNA end resection and provide insights into its role in HR mediated DSB repair. [ABSTRACT FROM AUTHOR]

The DNA damage response (DDR) comprises multiple functions that collectively preserve genomic integrity and suppress tumorigenesis. The Mre11 complex and ATM govern a major axis of the DDR and several lines of evidence implicate that axis in tumor suppression. Components of the Mre11 complex are mutated in approximately five percent of human cancers. Inherited mutations of complex members cause severe chromosome instability syndromes, such as Nijmegen Breakage Syndrome, which is associated with strong predisposition to malignancy. And in mice, Mre11 complex mutations are markedly more susceptible to oncogene- induced carcinogenesis. The complex is integral to all modes of DNA double strand break (DSB) repair and is required for the activation of ATM to effect DNA damage signaling. To understand which functions of the Mre11 complex are important for tumor suppression, we undertook mining of cancer genomic data from the clinical sequencing program at Memorial Sloan Kettering Cancer Center, which includes the Mre11 complex among the 468 genes assessed. Twenty five mutations in MRE11 and RAD50 were modeled in S. cerevisiae and in vitro. The mutations were chosen based on recurrence and conservation between human and yeast. We found that a significant fraction of tumor-borne RAD50 and MRE11 mutations exhibited separation of function phenotypes wherein Tel1/ATM activation was severely impaired while DNA repair functions were mildly or not affected. At the molecular level, the gene products of RAD50 mutations exhibited defects in ATP binding and hydrolysis. The data reflect the importance of Rad50 ATPase activity for Tel1/ATM activation and suggest that inactivation of ATM signaling confers an advantage to burgeoning tumor cells. Author summary: A complex network of functions is required for suppressing tumorigenesis. These include processes that regulate cell growth and differentiation, processes that repair damage to DNA and thereby prevent cancer promoting mutations and signaling pathways that lead to growth arrest and programmed cell death. The Mre11 complex influences both signaling and DNA repair. To understand its role in tumor suppression, we characterized mutations affecting members of the Mre11 complex that were uncovered through cancer genomic analyses. The data reveal that the signaling functions of the Mre11 complex are important for tumor suppression to a greater degree than its role in DNA repair. [ABSTRACT FROM AUTHOR]

Meiotic recombination is essential for faithful segregation of homologous chromosomes during gametogenesis. The progression of recombination is associated with dynamic changes in meiotic chromatin structures. However, whether Sycp2, a key structural component of meiotic chromatin, is required for the initiation of meiotic recombination is still unclear in vertebrates. Here, we describe that Sycp2 is required for assembly of the synaptonemal complex and early meiotic events in zebrafish spermatocytes. Our genetic screening by N-ethyl-N-nitrosourea mutagenesis revealed that ietsugu (its), a mutant zebrafish line with an aberrant splice site in the sycp2 gene, showed a defect during meiotic prophase I. The its mutation appeared to be a hypomorphic mutation compared to sycp2 knockout mutations generated by TALEN mutagenesis. Taking advantage of these sycp2 hypomorphic and knockout mutant lines, we demonstrated that Sycp2 is required for the assembly of the synaptonemal complex that is initiated in the vicinity of telomeres in wild-type zebrafish spermatocytes. Accordingly, homologous pairing, the foci of the meiotic recombinases Dmc1/Rad51 and RPA, and γH2AX signals were largely diminished in sycp2 knockout spermatocytes. Taken together, our data indicate that Sycp2 plays a critical role in not only the assembly of the synaptonemal complex, but also early meiotic recombination and homologous pairing, in vertebrates. Author summary: Meiosis is an essential type of cell division whose purpose is to produce gametes, such as eggs and sperm, for sexually reproducing eukaryotes. Most cells in the human body are diploid cells containing two homologous copies of each chromosome. Meiosis halves the genetic contents of diploid cells and produces haploid gametes with a single copy of each homologous chromosome pair. The faithful transmission of chromosomes is one of the main tasks in meiosis, since extra or missing chromosomes are common causes of genetic disorders, birth defects and infertility. Prior to proper segregation, homologs must pair up and be physically connected. This is achieved by homologous recombination initiated by programmed DNA double-strand breaks. Since it can be deleterious to cells, this initiation step is highly regulated by factors that are not fully understood. In this study, we found that Sycp2, a structural component of meiotic chromatin in metazoans, is essential for the initiation of homologous recombination, meiotic chromatin organization, and homologous pairing in zebrafish. These findings support recent biochemical studies that Sycp2 and Sycp3 serve as a vertebrate counterpart of S. cerevisiae Red1. [ABSTRACT FROM AUTHOR]

DNA double-strand breaks (DSBs) are toxic forms of DNA damage that must be repaired to maintain genome integrity. Telomerase can act upon a DSB to create a de novo telomere, a process that interferes with normal repair and creates terminal deletions. We previously identified sequences in Saccharomyces cerevisiae (SiRTAs; Sites of Repair-associated Telomere Addition) that undergo unusually high frequencies of de novo telomere addition, even when the original chromosome break is several kilobases distal to the eventual site of telomerase action. Association of the single-stranded telomere binding protein Cdc13 with a SiRTA is required to stimulate de novo telomere addition. Because extensive resection must occur prior to Cdc13 binding, we utilized these sites to monitor the effect of proteins involved in homologous recombination. We find that telomere addition is significantly reduced in the absence of the Rad51 recombinase, while loss of Rad52, required for Rad51 nucleoprotein filament formation, has no effect. Deletion of RAD52 suppresses the defect of the rad51Δ strain, suggesting that Rad52 inhibits de novo telomere addition in the absence of Rad51. The ability of Rad51 to counteract this effect of Rad52 does not require DNA binding by Rad51, but does require interaction between the two proteins, while the inhibitory effect of Rad52 depends on its interaction with Replication Protein A (RPA). Intriguingly, the genetic interactions we report between RAD51 and RAD52 are similar to those previously observed in the context of checkpoint adaptation. Forced recruitment of Cdc13 fully restores telomere addition in the absence of Rad51, suggesting that Rad52, through its interaction with RPA-coated single-stranded DNA, inhibits the ability of Cdc13 to bind and stimulate telomere addition. Loss of the Rad51-Rad52 interaction also stimulates a subset of Rad52-dependent microhomology-mediated repair (MHMR) events, consistent with the known ability of Rad51 to prevent single-strand annealing. Author summary: DNA double-strand breaks (DSBs) can lead to chromosome loss and rearrangement associated with cancer and genetic disease, so understanding how the cell coordinates multiple possible repair pathways is of critical importance. Telomerase is a ribonucleoprotein enzyme that uses an intrinsic RNA component as a template for the addition of highly repetitive, protective sequences (called telomeres) at normal chromosome ends. Rarely, telomerase acts upon a DSB to create a new or de novo telomere with resultant loss of sequences distal to the site of telomere addition. Here, we show that interactions between proteins with known roles during DSB repair modulate the probability of telomerase action at hotspots of de novo telomere addition in the yeast genome by influencing the association of Cdc13, a protein required for telomerase recruitment, with sites of telomere addition. Intriguingly, the same interactions that facilitate telomere addition prevent other types of rearrangements in response to chromosome breaks. [ABSTRACT FROM AUTHOR]

Extrachromosomal DNA can integrate into the genome with no sequence specificity producing an insertional mutation. This process, which is referred to as random integration (RI), requires a double stranded break (DSB) in the genome. Inducing DSBs by various means, including ionizing radiation, increases the frequency of integration. Here we report that non-lethal physiologically relevant doses of ionizing radiation (10–100 mGy), within the range produced by medical imaging equipment, stimulate RI of transfected and viral episomal DNA in human and mouse cells with an extremely high efficiency. Genetic analysis of the stimulated RI (S-RI) revealed that it is distinct from the background RI, requires histone H2AX S139 phosphorylation (γH2AX) and is not reduced by DNA polymerase θ (Polq) inactivation. S-RI efficiency was unaffected by the main DSB repair pathway (homologous recombination and non-homologous end joining) disruptions, but double deficiency in MDC1 and 53BP1 phenocopies γH2AX inactivation. The robust responsiveness of S-RI to physiological amounts of DSBs can be exploited for extremely sensitive, macroscopic and direct detection of DSB-induced mutations, and warrants further exploration in vivo to determine if the phenomenon has implications for radiation risk assessment. Author summary: Not all DNA in mammalian nuclei is organized into chromosomes. The pool of extrachromosomal DNA molecules is produced by natural process: during genomic DNA repair, viral infections, phagocytosis; and in experimental settings after transfection, and in gene therapy. Extrachromosomal DNA can integrate into the genome at the site of a double-stranded DNA break (DSB), producing a mutation in the chromosome. Because DSBs are dangerous lesions, they are actively eliminated, and their availability is a limiting factor for extrachromosomal DNA integration. It has long been known that inducing additional random DSBs, for example by exposing cells to ionizing radiation, can increase the frequency of random integration (RI), however the irradiation doses that were used were non-physiological. We found that much smaller doses of radiation, in the upper range of radiological diagnostic procedures, stimulate integration even more efficiently than the much larger doses studied previously. The potency of stimulation is remarkable, given that biological effects of such low doses are generally difficult to register, and warrants re-evaluation using animal models. Surprisingly, the genetic dependencies of radiation-stimulated integration do not include DSB repair pathways, and are distinct from background integration events. Our observations provide a hyper-sensitive tool to detect mutagenesis and reveal new information about the genetic interactions between DNA damage signaling and repair system components. [ABSTRACT FROM AUTHOR]

Recombinational repair of meiotic DNA double-strand breaks (DSBs) uses the homologous chromosome as a template, although the sister chromatid offers itself as a spatially more convenient substrate. In many organisms, this choice is reinforced by the recombination protein Dmc1. In Tetrahymena, the repair of DSBs, which are formed early in prophase, is postponed to late prophase when homologous chromosomes and sister chromatids become juxtaposed owing to tight parallel packing in the thread-shaped nucleus, and thus become equally suitable for use as repair templates. The delay in DSB repair is achieved by rejection of the invading strand by the Sgs1 helicase in early meiotic prophase. In the absence of Mcmd1, a meiosis-specific minichromosome maintenance (MCM)-like protein (and its partner Pamd1), Dmc1 is prematurely lost from chromatin and DNA synthesis (as monitored by BrdU incorporation) takes place in early prophase. In mcmd1Δ and pamd1Δ mutants, only a few crossovers are formed. In a mcmd1Δ hop2Δ double mutant, normal timing of Dmc1 loss and DNA synthesis is restored. Because Tetrahymena Hop2 is believed to enable homologous strand invasion, we conclude that Dmc1 loss in the absence of Mcmd1 affects only post-invasion recombination intermediates. Therefore, we propose that the Dmc1 nucleofilament becomes dismantled immediately after forming a heteroduplex with a template strand. As a consequence, repair synthesis and D-loop extension starts in early prophase intermediates and prevents strand rejection before the completion of homologous pairing. In this case, DSB repair may primarily use the sister chromatid. We conclude that Mcmd1‒Pamd1 protects the Dmc1 nucleofilament from premature dismantling, thereby suppressing precocious repair synthesis and excessive intersister strand exchange at the cost of homologous recombination. Author summary: Minichromosome maintenance (MCM) proteins are mainly known for their involvement in DNA replication. However, distant members of this protein family have recently been shown to promote interhomolog over intersister recombination in meiosis. They achieve this by enforcing or stabilizing the invasion of a double-stranded DNA by a filament consisting of a homologous single-stranded DNA molecule coated with a strand exchange protein. This interaction then would lead to the exchange of DNA strands and, ultimately, crossing over. Here, we study a member of the MCM protein family in the protist Tetrahymena thermophila. Meiosis in this organism has several unusual features: A synaptonemal complex is not formed, and homologous prealignment occurs during the close parallel arrangement of chromosomes in the extremely elongated, threadlike meiotic prophase nucleus. This noncanonical pairing has come along with altered mechanisms for recombination partner choice. Thus, we find that the Tetrahymena meiotic MCM protein promotes crossovers in an unprecedented way: It suppresses the formation of recombination intermediates between sister DNA molecules early in meiosis, thereby increasing the chance of competing interhomolog recombination events. Thus, members of the same protein family have been harnessed by different organisms to achieve the same result via completely different mechanisms. [ABSTRACT FROM AUTHOR]

During meiosis, homologous recombination repairs programmed DNA double-stranded breaks. Meiotic recombination physically links the homologous chromosomes ("homologs"), creating the tension between them that is required for their segregation. The central recombinase in this process is Dmc1. Dmc1's activity is regulated by its accessory factors including the heterodimeric protein Mei5-Sae3 and Rad51. We use a gain-of-function dmc1 mutant, dmc1-E157D, that bypasses Mei5-Sae3 to gain insight into the role of this accessory factor and its relationship to mitotic recombinase Rad51, which also functions as a Dmc1 accessory protein during meiosis. We find that Mei5-Sae3 has a role in filament formation and stability, but not in the bias of recombination partner choice that favors homolog over sister chromatids. Analysis of meiotic recombination intermediates suggests that Mei5-Sae3 and Rad51 function independently in promoting filament stability. In spite of its ability to load onto single-stranded DNA and carry out recombination in the absence of Mei5-Sae3, recombination promoted by the Dmc1 mutant is abnormal in that it forms foci in the absence of DNA breaks, displays unusually high levels of multi-chromatid and intersister joint molecule intermediates, as well as high levels of ectopic recombination products. We use super-resolution microscopy to show that the mutant protein forms longer foci than those formed by wild-type Dmc1. Our data support a model in which longer filaments are more prone to engage in aberrant recombination events, suggesting that filament lengths are normally limited by a regulatory mechanism that functions to prevent recombination-mediated genome rearrangements. Author summary: During meiosis, two rounds of division follow a single round of DNA replication to create the gametes for biparental reproduction. The first round of division requires that the homologous chromosomes become physically linked to one another to create the tension that is necessary for their segregation. This linkage is achieved through DNA recombination between the two homologous chromosomes, followed by resolution of the recombination intermediate into a crossover. Central to this process is the meiosis-specific recombinase Dmc1, and its accessory factors, which provide important regulatory functions to ensure that recombination is accurate, efficient, and occurs predominantly between homologous chromosomes, and not sister chromatids. To gain insight into the regulation of Dmc1 by its accessory factors, we mutated Dmc1 such that it was no longer dependent on its accessory factor Mei5-Sae3. Our analysis reveals that Dmc1 accessory factors Mei5-Sae3 and Rad51 have independent roles in stabilizing Dmc1 filaments. Furthermore, we find that although Rad51 is required for promoting recombination between homologous chromosomes, Mei5-Sae3 is not. Lastly, we show that our Dmc1 mutant forms abnormally long filaments, and high levels of aberrant recombination intermediates and products. These findings suggest that filaments are actively maintained at short lengths to prevent deleterious genome rearrangements. [ABSTRACT FROM AUTHOR]