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DNA ligase (LIG) I and IIIα finalize base excision repair (BER) by sealing a nick product after nucleotide insertion by DNA polymerase (pol) β at the downstream steps. We previously demonstrated that a functional interplay between polβ and BER ligases is critical for efficient repair, and polβ mismatch or oxidized nucleotide insertions confound the final ligation step. Yet, how targeting downstream enzymes with small molecule inhibitors could affect this coordination remains unknown. Here, we report that DNA ligase inhibitors, L67 and L82-G17, slightly enhance hypersensitivity to oxidative stress-inducing agent, KBrO3, in polβ+/+ cells more than polβ-/- null cells. We showed less efficient ligation after polβ nucleotide insertions in the presence of the DNA ligase inhibitors. Furthermore, the mutations at the ligase inhibitor binding sites (G448, R451, A455) of LIG1 significantly affect nick DNA binding affinity and nick sealing efficiency. Finally, our results demonstrated that the BER ligases seal a gap repair intermediate by the effect of polβ inhibitor that diminishes gap filling activity. Overall, our results contribute to understand how the BER inhibitors against downstream enzymes, polβ, LIG1, and LIGIIIα, could impact the efficiency of gap filling and subsequent nick sealing at the final steps leading to the formation of deleterious repair intermediates. [ABSTRACT FROM AUTHOR]

The recent discovery of non-proteinaceous ubiquitylation substrates broadened our understanding of this modification beyond conventional protein targets. However, the existence of additional types of substrates remains elusive. Here, we present evidence that nucleic acids can also be directly ubiquitylated via ester bond formation. DTX3L, a member of the DELTEX family E3 ubiquitin ligases, ubiquitylates DNA and RNA in vitro and that this activity is shared with DTX3, but not with the other DELTEX family members DTX1, DTX2 and DTX4. DTX3L shows preference for the 3′-terminal adenosine over other nucleotides. In addition, we demonstrate that ubiquitylation of nucleic acids is reversible by DUBs such as USP2, JOSD1 and SARS-CoV-2 PLpro. Overall, our study proposes reversible ubiquitylation of nucleic acids in vitro and discusses its potential functional implications. Synopsis: Nucleic acids are a novel type of ubiquitylation substrate. Nucleic acids are ubiquitylated by DELTEX ubiquitin E3 ligase DTX3L, targeting the 3′-terminal adenosine nucleotides at their 3′ hydroxyl groups. KH domain-containing DELTEX E3 ligase DTX3 and DTX3L possess nucleic acids ubiquitylation activity while WWE domain-containing DELTEX E3 ligase DTX1, DTX2 as well as DTX4 do not. Ubiquitylation of nucleic acids by DTX3L could block cleavage by 3′–5′ nuclease and represents a reversible process. Nucleic acids are a novel type of ubiquitylation substrate. [ABSTRACT FROM AUTHOR]

Targeting DNA repair pathways is an important strategy in anticancer therapy. However, the unrevealed interactions between different DNA repair systems may interfere with the desired therapeutic effect. Among DNA repair systems, BER and NHEJ protect genome integrity through the entire cell cycle. BER is involved in the repair of DNA base lesions and DNA single-strand breaks (SSBs), while NHEJ is responsible for the repair of DNA double-strand breaks (DSBs). Previously, we showed that BER deficiency leads to downregulation of NHEJ gene expression. Here, we studied BER's response to NHEJ deficiency induced by knockdown of NHEJ scaffold protein XRCC4 and compared the knockdown effects in normal (TIG-1) and hTERT-modified cells (NBE1). We investigated the expression of the XRCC1, LIG3, and APE1 genes of BER and LIG4; the Ku70/Ku80 genes of NHEJ at the mRNA and protein levels; as well as p53, Sp1 and PARP1. We found that, in both cell lines, XRCC4 knockdown leads to a decrease in the mRNA levels of both BER and NHEJ genes, though the effect on protein level is not uniform. XRCC4 knockdown caused an increase in p53 and Sp1 proteins, but caused G1/S delay only in normal cells. Despite the increased p53 protein, p21 did not significantly increase in NBE1 cells with overexpressed hTERT, and this correlated with the absence of G1/S delay in these cells. The data highlight the regulatory function of the XRCC4 scaffold protein and imply its connection to a transcriptional regulatory network or mRNA metabolism. [ABSTRACT FROM AUTHOR]

Single-strand DNA-binding proteins SSB/RPA are ubiquitous and essential proteins that bind ssDNA in bacteria/eukaryotes and coordinate DNA metabolic processes such as replication, repair, and recombination. SSB protects ssDNA from degradation by nucleases, while also facilitating/regulating the activity of multiple partner proteins involved in DNA processes. Using Spi− assay, which detects aberrantly excised λ prophage from the E. coli chromosome as a measure of illegitimate recombination (IR) occurrence, we have shown that SSB inhibits IR in several DSB resection pathways. The conditional ssb-1 mutation produced a higher IR increase at the nonpermissive temperature than the recQ inactivation. A double ssb-1 recQ mutant had an even higher level of IR, while showing reduced homologous recombination (HR). Remarkably, the ssb gene overexpression complemented recQ deficiency in suppressing IR, indicating that the SSB function is epistatic to RecQ. Overproduced truncated SSBΔC8 protein, which binds to ssDNA, but does not interact with partner proteins, only partially complemented recQ and ssb-1 mutations, while causing an IR increase in otherwise wild-type bacteria, suggesting that ssDNA binding of SSB is required but not sufficient for effective IR inhibition, which rather entails interaction with RecQ and likely some other protein(s). Our results depict SSB as the main genome caretaker in E. coli, which facilitates HR while inhibiting IR. In enabling high-fidelity DSB repair under physiological conditions SSB is assisted by RecQ helicase, whose activity it controls. Conversely, an excess of SSB renders RecQ redundant for IR suppression. [ABSTRACT FROM AUTHOR]

Abstract Single-strand DNA-binding proteins SSB/RPA are ubiquitous and essential proteins that bind ssDNA in bacteria/eukaryotes and coordinate DNA metabolic processes such as replication, repair, and recombination. SSB protects ssDNA from degradation by nucleases, while also facilitating/regulating the activity of multiple partner proteins involved in DNA processes. Using Spi− assay, which detects aberrantly excised λ prophage from the E. coli chromosome as a measure of illegitimate recombination (IR) occurrence, we have shown that SSB inhibits IR in several DSB resection pathways. The conditional ssb-1 mutation produced a higher IR increase at the nonpermissive temperature than the recQ inactivation. A double ssb-1 recQ mutant had an even higher level of IR, while showing reduced homologous recombination (HR). Remarkably, the ssb gene overexpression complemented recQ deficiency in suppressing IR, indicating that the SSB function is epistatic to RecQ. Overproduced truncated SSBΔC8 protein, which binds to ssDNA, but does not interact with partner proteins, only partially complemented recQ and ssb-1 mutations, while causing an IR increase in otherwise wild-type bacteria, suggesting that ssDNA binding of SSB is required but not sufficient for effective IR inhibition, which rather entails interaction with RecQ and likely some other protein(s). Our results depict SSB as the main genome caretaker in E. coli, which facilitates HR while inhibiting IR. In enabling high-fidelity DSB repair under physiological conditions SSB is assisted by RecQ helicase, whose activity it controls. Conversely, an excess of SSB renders RecQ redundant for IR suppression.

Transcription is a major contributor to genomic instability. The ribosomal RNA (rDNA) gene locus consists of a head-to-tail repeat of the most actively transcribed genes in the genome. RNA polymerase I (RNAPI) is responsible for massive rRNA production, and nascent rRNA is co-transcriptionally assembled with early assembly factors in the yeast nucleolus. In Saccharomyces cerevisiae, a mutant form of RNAPI bearing a fusion of the transcription factor Rrn3 with RNAPI subunit Rpa43 (CARA-RNAPI) has been described previously. Here, we show that the CARA-RNAPI allele results in a novel type of rRNA processing defect, associated with rDNA genomic instability. A fraction of the 35S rRNA produced in CARA-RNAPI mutant escapes processing steps and accumulates. This accumulation is increased in mutants affecting exonucleolytic activities of the exosome complex. CARA-RNAPI is synthetic lethal with monopolin mutants that are known to affect the rDNA condensation. CARA-RNAPI strongly impacts rDNA organization and increases rDNA copy number variation. Reduced rDNA copy number suppresses lethality, suggesting that the chromosome segregation defect is caused by genomic rDNA instability. We conclude that a constitutive association of Rrn3 with transcribing RNAPI results in the accumulation of rRNAs that escape normal processing, impacting rDNA organization and affecting rDNA stability.

Abstract Background Enhancing crops’ drought resilience is necessary to maintain productivity levels. Plants interact synergistically with microorganisms like Beauveria bassiana to improve drought tolerance. Therefore, the current study investigates the effects of biopriming with B. bassiana on drought tolerance in Malva parviflora plants grown under regular irrigation (90% water holding capacity (WHC)), mild (60% WHC), and severe drought stress (30% WHC). Results The results showed that drought stress reduced the growth and physiological attributes of M. parviflora. However, those bioprimed with B. bassiana showed higher drought tolerance and enhanced growth, physiological, and biochemical parameters: drought stress enriched malondialdehyde and H2O2 contents. Conversely, exposure to B. bassiana reduced stress markers and significantly increased proline and ascorbic acid content under severe drought stress; it enhanced gibberellic acid and reduced ethylene. Bioprimed M. parviflora, under drought conditions, improved antioxidant enzymatic activity and the plant’s nutritional status. Besides, ten Inter-Simple Sequence Repeat primers detected a 25% genetic variation between treatments. Genomic DNA template stability (GTS) decreased slightly and was more noticeable in response to drought stress; however, for drought-stressed plants, biopriming with B. bassiana retained the GTS. Conclusion Under drought conditions, biopriming with B. bassiana enhanced Malva’s growth and nutritional value. This could attenuate photosynthetic alterations, up-regulate secondary metabolites, activate the antioxidant system, and maintain genome integrity.

Abstract Genome replication is frequently impeded by highly stable DNA secondary structures, including G-quadruplex (G4) DNA, that can hinder the progression of the replication fork. Human WRNIP1 (Werner helicase Interacting Protein 1) associates with various components of the replication machinery and plays a crucial role in genome maintenance processes. However, its detailed function is still not fully understood. Here we show that human WRNIP1 interacts with G4 structures and provide evidence for its contribution to G4 processing. The absence of WRNIP1 results in elevated levels of G4 structures, DNA damage and chromosome aberrations following treatment with PhenDC3, a G4-stabilizing ligand. Additionally, we establish a functional and physical relationship between WRNIP1 and the PIF1 helicase in G4 processing. In summary, our results suggest that WRNIP1 aids genome replication and maintenance by regulating G4 processing and this activity relies on Pif1 DNA helicase.

SUMMARY: DNA–protein crosslinks (DPCs) are highly toxic DNA lesions represented by proteins covalently bound to the DNA. Persisting DPCs interfere with fundamental genetic processes such as DNA replication and transcription. Cytidine analog zebularine (ZEB) has been shown to crosslink DNA METHYLTRANSFERASE1 (MET1). Recently, we uncovered a critical role of the SMC5/6‐mediated SUMOylation in the repair of DPCs. In an ongoing genetic screen, we identified two additional candidates, HYPERSENSITIVE TO ZEBULARINE 2 and 3, that were mapped to REGULATOR OF TELOMERE ELONGATION 1 (RTEL1) and polymerase TEBICHI (TEB), respectively. By monitoring the growth of hze2 and hze3 plants in response to zebularine, we show the importance of homologous recombination (HR) factor RTEL1 and microhomology‐mediated end‐joining (MMEJ) polymerase TEB in the repair of MET1‐DPCs. Moreover, genetic interaction and sensitivity assays showed the interdependency of SMC5/6 complex, HR, and MMEJ in the homology‐directed repair of MET1‐DPCs in Arabidopsis. Altogether, we provide evidence that MET1‐DPC repair in plants is more complex than originally expected. Significance Statement: DNA–protein crosslinks (DPCs) are highly toxic DNA lesions represented by proteins covalently bound to the DNA. We show the importance of homologous recombination factor RTEL1 and microhomology‐mediated end joining polymerase TEB and their interdependency with the SMC5/6 complex in the repair of DNA METHYLTRANSFERASE1‐DPCs. This suggests a complex network of pathways detoxifying DPCs in plants. [ABSTRACT FROM AUTHOR]

MCM8 has emerged as a core gene in reproductive aging and is crucial for meiotic homologous recombination repair. It also safeguards genome stability by coordinating the replication stress response during mitosis, but its function in mitotic germ cells remains elusive. Here we found that disabling MCM8 in mice resulted in proliferation defects of primordial germ cells (PGCs) and ultimately impaired fertility. We further demonstrated that MCM8 interacted with two known helicases DDX5 and DHX9, and loss of MCM8 led to R-loop accumulation by reducing the retention of these helicases at R-loops, thus inducing genome instability. Cells expressing premature ovarian insufficiency-causative mutants of MCM8 with decreased interaction with DDX5 displayed increased R-loop levels. These results show MCM8 interacts with R-loop-resolving factors to prevent R-loop-induced DNA damage, which may contribute to the maintenance of genome integrity of PGCs and reproductive reserve establishment. Our findings thus reveal an essential role for MCM8 in PGC development and improve our understanding of reproductive aging caused by genome instability in mitotic germ cells. Synopsis: MCM8 deficiency impairs meiotic homologous recombination repair, but the specific role of MCM8 in mitotic germ cells remains poorly understood. This study reveals that MCM8 interacts with helicase DDX5 to prevent R-loop-induced DNA damage, and maintains primordial germ cell (PGC) genome stability and rapid proliferation. MCM8 safeguards PGC rapid proliferation to establish reproductive reserve. MCM8 retains DDX5 at R-loops to prevent R-loop-induced DNA damage. MCM8 mutations associated with premature ovarian insufficiency (POI) affect interactions with DDX5 and increase R-loop levels. Loss of MCM8 compromises primordial germ cell proliferation in mice, possibly via DNA damage caused by reduced retention of R-loop-resolving factors. [ABSTRACT FROM AUTHOR]

Genome replication is frequently impeded by highly stable DNA secondary structures, including G-quadruplex (G4) DNA, that can hinder the progression of the replication fork. Human WRNIP1 (Werner helicase Interacting Protein 1) associates with various components of the replication machinery and plays a crucial role in genome maintenance processes. However, its detailed function is still not fully understood. Here we show that human WRNIP1 interacts with G4 structures and provide evidence for its contribution to G4 processing. The absence of WRNIP1 results in elevated levels of G4 structures, DNA damage and chromosome aberrations following treatment with PhenDC3, a G4-stabilizing ligand. Additionally, we establish a functional and physical relationship between WRNIP1 and the PIF1 helicase in G4 processing. In summary, our results suggest that WRNIP1 aids genome replication and maintenance by regulating G4 processing and this activity relies on Pif1 DNA helicase. [ABSTRACT FROM AUTHOR]

In fission yeast lacking the telomere binding protein, Taz1, replication forks stall at telomeres, triggering deleterious downstream events. Strand invasion from one taz1Δ telomeric stalled fork to another on a separate (nonsister) chromosome leads to telomere entanglements, which are resolved in mitosis at 32°C; however, entanglement resolution fails at ≤20°C, leading to cold-specific lethality. Previously, we found that loss of the mitotic function of Rif1, a conserved DNA replication and repair factor, suppresses cold sensitivity by promoting resolution of entanglements without affecting entanglement formation. To understand the underlying pathways of mitotic entanglement resolution, we performed a series of genome-wide synthetic genetic array screens to generate a comprehensive list of genetic interactors of taz1 Δ and rif1 Δ. We modified a previously described screening method to ensure that the queried cells were kept in log phase growth. In addition to recapitulating previously identified genetic interactions, we find that loss of genes encoding components of the nuclear pore complex (NPC) promotes telomere disentanglement and suppresses taz1Δ cold sensitivity. We attribute this to more rapid anaphase midregion nuclear envelope (NE) breakdown in the absence of these NPC components. Loss of genes involved in lipid metabolism reverses the ability of rif1 + deletion to suppress taz1Δ cold sensitivity, again pinpointing NE modulation. A rif1 + separation-of-function mutant that specifically loses Rif1's mitotic functions yields similar genetic interactions. Genes promoting membrane fluidity were enriched in a parallel taz1+ synthetic lethal screen at permissive temperature, cementing the idea that the cold specificity of taz1Δ lethality stems from altered NE homeostasis. [ABSTRACT FROM AUTHOR]

The nucleoside analogue decitabine (or 5-aza-dC) is used to treat several haematological cancers. Upon its triphosphorylation and incorporation into DNA, 5-aza-dC induces covalent DNA methyltransferase 1 DNA–protein crosslinks (DNMT1-DPCs), leading to DNA hypomethylation. However, 5-aza-dC's clinical outcomes vary, and relapse is common. Using genome-scale CRISPR/Cas9 screens, we map factors determining 5-aza-dC sensitivity. Unexpectedly, we find that loss of the dCMP deaminase DCTD causes 5-aza-dC resistance, suggesting that 5-aza-dUMP generation is cytotoxic. Combining results from a subsequent genetic screen in DCTD-deficient cells with the identification of the DNMT1-DPC-proximal proteome, we uncover the ubiquitin and SUMO1 E3 ligase, TOPORS, as a new DPC repair factor. TOPORS is recruited to SUMOylated DNMT1-DPCs and promotes their degradation. Our study suggests that 5-aza-dC-induced DPCs cause cytotoxicity when DPC repair is compromised, while cytotoxicity in wild-type cells arises from perturbed nucleotide metabolism, potentially laying the foundations for future identification of predictive biomarkers for decitabine treatment. Synopsis: The chemotherapeutic decitabine/5-aza-dC induces covalent DNMT1 DNA-protein crosslinks (DPCs), leading to DNMT1 degradation and DNA hypomethylation. This study finds that E3 ligase TOPORS promotes DNMT1-DPC degradation, while deaminase DCTD mediates a DNMT1-independent mode of 5-aza-dC cytotoxicity. The dCMP deaminase DCTD mediates 5-aza-dC cytotoxicity independently of DNA-protein crosslink formation, through the generation of 5-aza-dUMP. The ubiquitin and SUMO1 E3 ligase TOPORS is a novel player in global-genome DPC repair. TOPORS is recruited to SUMOylated DNMT1-DPCs through its SUMO-interaction motifs, whereupon it promotes DNMT1-DPC ubiquitylation and degradation. TOPORS acts in parallel to the E3 ubiquitin ligase RNF4 in mediating DNMT1-DPC tolerance. TOPORS acts in parallel to the ubiquitin ligase RNF4 in degradation of DNMT1-DNA crosslinks, thereby mediating cellular tolerance to these DPCs. [ABSTRACT FROM AUTHOR]

p53 was discovered 45 years ago as an SV40 large T antigen binding protein, coded by the most frequently mutated TP53 gene in human cancers. As a transcription factor, p53 is tightly regulated by a rich network of post-translational modifications to execute its diverse functions in tumor suppression. Although early studies established p53-mediated cell-cycle arrest, apoptosis, and senescence as the classic barriers in cancer development, a growing number of new functions of p53 have been discovered and the scope of p53-mediated anti-tumor activity is largely expanded. Here, we review the complexity of different layers of p53 regulation, and the recent advance of the p53 pathway in metabolism, ferroptosis, immunity, and others that contribute to tumor suppression. We also discuss the challenge regarding how to activate p53 function specifically effective in inhibiting tumor growth without harming normal homeostasis for cancer therapy. p53 plays a central role in suppressing tumorigenesis. In this issue, Liu et al. review the complexity of different layers of p53 regulation, and the recent advance of the p53 pathway in metabolism, ferroptosis, immunity, and others that largely expand the scope of its anti-tumor activity. [ABSTRACT FROM AUTHOR]

The ribosomal DNA locus (rDNA) is central for the functioning of cells because it encodes ribosomal RNAs, key components of ribosomes, and also because of its links to fundamental metabolic processes, with significant impact on genome integrity and aging. The repetitive nature of the rDNA gene units forces the locus to maintain sequence homogeneity through recombination processes that are closely related to genomic stability. The co-presence of basic DNA transactions, such as replication, transcription by major RNA polymerases, and recombination, in a defined and restricted area of the genome is of particular relevance as it affects the stability of the rDNA locus by both direct and indirect mechanisms. This condition is well exemplified by the rDNA of Saccharomyces cerevisiae. In this review we summarize essential knowledge on how the complexity and overlap of different processes contribute to the control of rDNA and genomic stability in this model organism. [ABSTRACT FROM AUTHOR]

Dynamic nuclear architecture and chromatin organizations are the key features of the mid‐prophase I in mammalian meiosis. The chromatin undergoes major changes, including meiosis‐specific spatiotemporal arrangements and remodeling, the establishment of chromatin loop–axis structure, pairing, and crossing over between homologous chromosomes, any deficiencies in these events may induce genome instability, subsequently leading to failure to produce gametes and infertility. Despite the significance of chromatin structure, little is known about the location of chromatin marks and the necessity of their balance during meiosis prophase I. Here, we show a thorough cytological study of the surface‐spread meiotic chromosomes of mouse spermatocytes for H3K9,14,18,23,27,36, H4K12,16 acetylation, and H3K4,9,27,36 methylation. Active acetylation and methylation marks on H3 and H4, such as H3K9ac, H3K14ac, H3K18ac, H3K36ac, H3K56ac, H4K12ac, H4K16ac, and H3K36me3 exhibited pan‐nuclear localization away from heterochromatin. In comparison, repressive marks like H3K9me3 and H3K27me3 are localized to heterochromatin. Further, taking advantage of the delivery of small‐molecule chemical inhibitors methotrexate (heterochromatin enhancer), heterochromatin inhibitor, anacardic acid (histone acetyltransferase inhibitor), trichostatin A (histone deacetylase inhibitor), IOX1 (JmjC demethylases inhibitor), and AZ505 (methyltransferase inhibitor) in seminiferous tubules through the rete testis route, revealed that alteration in histone modifications enhanced the centromere mislocalization, chromosome breakage, altered meiotic recombination and reduced sperm count. Specifically, IOX1 and AZ505 treatment shows severe meiotic phenotypes, including altering chromosome axis length and chromatin loop size via transcriptional regulation of meiosis‐specific genes. Our findings highlight the importance of balanced chromatin modifications in meiotic prophase I chromosome organization and instability. [ABSTRACT FROM AUTHOR]

Nypa fruticans (Wurmb), a mangrove palm species with origins dating back to the Late Cretaceous period, is a unique species for investigating long‐term adaptation strategies to intertidal environments and the early evolution of palms. Here, we present a chromosome‐level genome sequence and assembly for N. fruticans. We integrated the genomes of N. fruticans and other palm family members for a comparative genomic analysis, which confirmed that the common ancestor of all palms experienced a whole‐genome duplication event around 89 million years ago, shaping the distinctive characteristics observed in this clade. We also inferred a low mutation rate for the N. fruticans genome, which underwent strong purifying selection and evolved slowly, thus contributing to its stability over a long evolutionary period. Moreover, ancient duplicates were preferentially retained, with critical genes having experienced positive selection, enhancing waterlogging tolerance in N. fruticans. Furthermore, we discovered that the pseudogenization of Early Methionine‐labelled 1 (EM1) and EM6 in N. fruticans underly its crypto‐vivipary characteristics, reflecting its intertidal adaptation. Our study provides valuable genomic insights into the evolutionary history, genome stability, and adaptive evolution of the mangrove palm. Our results also shed light on the long‐term adaptation of this species and contribute to our understanding of the evolutionary dynamics in the palm family. [ABSTRACT FROM AUTHOR]

DNA damage occurs throughout life from a variety of sources, and it is imperative to repair damage in a timely manner to maintain genome stability. Thus, DNA repair mechanisms are a fundamental part of life. Nucleotide excision repair (NER) plays an important role in the removal of bulky DNA adducts, such as cyclobutane pyrimidine dimers from ultraviolet light or DNA crosslinking damage from platinum‐based chemotherapeutics, such as cisplatin. A main component for the NER pathway is transcription factor IIH (TFIIH), a multifunctional, 10‐subunit protein complex with crucial roles in both transcription and NER. In transcription, TFIIH is a component of the pre‐initiation complex and is important for promoter opening and the phosphorylation of RNA Polymerase II (RNA Pol II). During repair, TFIIH is important for DNA unwinding, recruitment of downstream repair factors, and verification of the bulky lesion. Several different disease states can arise from mutations within subunits of the TFIIH complex. Most strikingly are xeroderma pigmentosum (XP), XP combined with Cockayne syndrome (CS), and trichothiodystrophy (TTD). Here, we summarize the recruitment and functions of TFIIH in the two NER subpathways, global genomic (GG‐NER) and transcription‐coupled NER (TC‐NER). We will also discuss how TFIIH's roles in the two subpathways lead to different genetic disorders. [ABSTRACT FROM AUTHOR]

Although both are salient features of genomes, at first glance ribosomal DNAs and transposable elements are genetic elements with not much in common: whereas ribosomal DNAs are mainly viewed as housekeeping genes that uphold all prime genome functions, transposable elements are generally portrayed as selfish and disruptive. These opposing characteristics are also mirrored in other attributes: organization in tandem (ribosomal DNAs) versus organization in a dispersed manner (transposable elements); evolution in a concerted manner (ribosomal DNAs) versus evolution by diversification (transposable elements); and activity that prolongs genomic stability (ribosomal DNAs) versus activity that shortens it (transposable elements). Re-visiting relevant instances in which ribosomal DNA–transposable element interactions have been reported, we note that both repeat types share at least four structural and functional hallmarks: (1) they are repetitive DNAs that shape genomes in evolutionary timescales, (2) they exchange structural motifs and can enter co-evolution processes, (3) they are tightly controlled genomic stress sensors playing key roles in senescence/aging, and (4) they share common epigenetic marks such as DNA methylation and histone modification. Here, we give an overview of the structural, functional, and evolutionary characteristics of both ribosomal DNAs and transposable elements, discuss their roles and interactions, and highlight trends and future directions as we move forward in understanding ribosomal DNA–transposable element associations. [ABSTRACT FROM AUTHOR]

The RNA-binding protein LUC7L3 is the human homolog of yeast U1 small nuclear RNA (snRNA)-related splicing factor Luc7p. While the primary function of LUC7L3 as an RNA-binding protein is believed to be involved in RNA metabolism, particularly in the splicing process, its exact role and other functions are still not fully understood. In this study, we aimed to elucidate the role of LUC7L3 and its impact on cell proliferation. Our study revealed that LUC7L3 depletion impairs cell proliferation compared to the other Luc7p paralogs, resulting in cell apoptosis and senescence. We explored the underlying mechanisms and found that LUC7L3 depletion leads to R-loop accumulation, DNA replication stress, and genome instability. Furthermore, we discovered that LUC7L3 depletion caused abnormalities in spindle assembly, leading to the formation of multinuclear cells. This was attributed to the dysregulation of protein translation of spindle-associated proteins. Additionally, we investigated the interplay between LUC7L3 and SRSF1 and identified SRSF1 as an upper stream regulator of LUC7L3, promoting the translation of LUC7L3 protein. These findings highlight the importance of LUC7L3 in maintaining genome stability and its relationship with SRSF1 in this regulatory pathway.

Targeting DNA repair pathways is an important strategy in anticancer therapy. However, the unrevealed interactions between different DNA repair systems may interfere with the desired therapeutic effect. Among DNA repair systems, BER and NHEJ protect genome integrity through the entire cell cycle. BER is involved in the repair of DNA base lesions and DNA single-strand breaks (SSBs), while NHEJ is responsible for the repair of DNA double-strand breaks (DSBs). Previously, we showed that BER deficiency leads to downregulation of NHEJ gene expression. Here, we studied BER’s response to NHEJ deficiency induced by knockdown of NHEJ scaffold protein XRCC4 and compared the knockdown effects in normal (TIG-1) and hTERT-modified cells (NBE1). We investigated the expression of the XRCC1, LIG3, and APE1 genes of BER and LIG4; the Ku70/Ku80 genes of NHEJ at the mRNA and protein levels; as well as p53, Sp1 and PARP1. We found that, in both cell lines, XRCC4 knockdown leads to a decrease in the mRNA levels of both BER and NHEJ genes, though the effect on protein level is not uniform. XRCC4 knockdown caused an increase in p53 and Sp1 proteins, but caused G1/S delay only in normal cells. Despite the increased p53 protein, p21 did not significantly increase in NBE1 cells with overexpressed hTERT, and this correlated with the absence of G1/S delay in these cells. The data highlight the regulatory function of the XRCC4 scaffold protein and imply its connection to a transcriptional regulatory network or mRNA metabolism.

Uncontrolled pathogenic genome mutations in germline cells might impair adult fertility, lead to birth defects or even affect the adaptability of a species. Understanding the sources of DNA damage, as well as the features of damage response in germline cells are the overarching tasks to reduce the mutations in germline cells. With the accumulation of human genome data and genetic reports, genome variants formed in germline cells are being extensively explored. However, the sources of DNA damage, the damage repair mechanisms, and the effects of DNA damage or mutations on the development of germline cells are still unclear. Besides exogenous triggers of DNA damage such as irradiation and genotoxic chemicals, endogenous exposure to inflammation may also contribute to the genome instability of germline cells. In this review, we summarized the features of de novo mutations and the specific DNA damage responses in germline cells and explored the possible roles of inflammation on the genome stability of germline cells. [ABSTRACT FROM AUTHOR]

FANCJ, a DNA helicase linked to Fanconi anemia and frequently mutated in cancers, counteracts replication stress by dismantling unconventional DNA secondary structures (such as G-quadruplexes) that occur at the DNA replication fork in certain sequence contexts. However, how FANCJ is recruited to the replisome is unknown. Here, we report that FANCJ directly binds to AND-1 (the vertebrate ortholog of budding yeast Ctf4), a homo-trimeric protein adaptor that connects the CDC45/MCM2-7/GINS replicative DNA helicase with DNA polymerase α and several other factors at DNA replication forks. The interaction between FANCJ and AND-1 requires the integrity of an evolutionarily conserved Ctf4-interacting protein (CIP) box located between the FANCJ helicase motifs IV and V. Disruption of the CIP box significantly reduces FANCJ association with the replisome, causing enhanced DNA damage, decreased replication fork recovery and fork asymmetry in cells unchallenged or treated with Pyridostatin, a G-quadruplex-binder, or Mitomycin C, a DNA inter-strand cross-linking agent. Cancer-relevant FANCJ CIP box variants display reduced AND-1-binding and enhanced DNA damage, a finding that suggests their potential role in cancer predisposition. Synopsis: FANCJ/BRIP1, a DNA helicase counteracting replication stress, is recruited to the DNA replication machinery by directly binding to AND-1. Disruption of the FANCJ/AND-1 interaction causes enhanced DNA damage, replication fork stalling and asymmetry in either unperturbed or stressful conditions. Human FANCJ associates with the DNA replication machinery through an interaction with AND-1 that is mediated by a Ctf4-interacting protein (CIP) box. The FANCJ/AND-1 interaction is important for promoting replication fork progression and suppressing DNA damage. Previously uncharacterized FANCJ CIP box missense mutations, found in cancer patients, reduce AND-1-binding and enhance DNA damage. FANCJ/BRIP1, a DNA helicase counteracting replication stress, is recruited to the DNA replication machinery by directly binding to AND-1. Disruption of the FANCJ/AND-1 interaction causes enhanced DNA damage, replication fork stalling and asymmetry in either unperturbed or stressful conditions. [ABSTRACT FROM AUTHOR]

Mammalian telomeres have evolved safeguards to prevent their recognition as DNA double‐stranded breaks by suppressing the activation of various DNA sensing and repair proteins. We have shown that the telomere‐binding proteins TRF2 and RAP1 cooperate to prevent telomeres from undergoing aberrant homology‐directed recombination by mediating t‐loop protection. Our recent findings also suggest that mammalian telomere‐binding proteins interact with the nuclear envelope to maintain chromosome stability. RAP1 interacts with nuclear lamins through KU70/KU80, and disruption of RAP1 and TRF2 function result in nuclear envelope rupture, promoting telomere‐telomere recombination to form structures termed ultrabright telomeres. In this review, we discuss the importance of the interactions between shelterin components and the nuclear envelope to maintain telomere homeostasis and genome stability. [ABSTRACT FROM AUTHOR]

Blackcurrant (Ribes nigrum L.) is a classical fruit that has long been used to make juice, jam, and liqueur. Blackcurrant extract is known to relieve cells from DNA damage caused by hydrogen peroxide (H2O2), methyl methane sulfonate (MMS), and ultraviolet (UV) radiation. We found that blackcurrant extract (BCE) stabilizes the ribosomal RNA gene cluster (rDNA), one of the most unstable regions in the genome, through repression of noncoding transcription in the intergenic spacer (IGS) which extended the lifespan in budding yeast. Reduced formation of extrachromosomal circles (ERCs) after exposure to fractionated BCE suggested that acidity of the growth medium impacted rDNA stability. Indeed, alteration of the acidity of the growth medium to pH ~4.5 by adding HCl increased rDNA stability and extended the lifespan. We identified RPD3 as the gene responsible for this change, which was mediated by the RPD3L histone deacetylase complex. In mammals, as inflammation sites in a tissue are acidic, DNA maintenance may be similarly regulated to prevent genome instability from causing cancer. [ABSTRACT FROM AUTHOR]

Germline RTEL1 mutations cause diverse clinical conditions in telomere biology diseases. RTEL1 interacts with various proteins and nucleic acids, and unwinds various structures, including R-loops and G-quadruplexes. RTEL1 functions in different nuclear pathways as a helicase and possibly also independently of its helicase activity. RTEL1 is key to understanding the interplay between DNA replication and repair, transcription, and telomere maintenance, as well as their implications for cell proliferation, aging, and cancer. Regulator of telomere elongation 1 (RTEL1) is known as a DNA helicase that is important for telomeres and genome integrity. However, the diverse phenotypes of RTEL1 dysfunction, the wide spectrum of symptoms caused by germline RTEL1 mutations, and the association of RTEL1 mutations with cancers suggest that RTEL1 is a complex machine that interacts with DNA, RNA, and proteins, and functions in diverse cellular pathways. We summarize the proposed functions of RTEL1 and discuss their implications for telomere maintenance. Studying RTEL1 is crucial for understanding the complex interplay between telomere maintenance and other nuclear pathways, and how compromising these pathways causes telomere biology diseases, various aging-associated pathologies, and cancer. [ABSTRACT FROM AUTHOR]

The RNA/DNA helicase senataxin (SETX) has been involved in multiple crucial processes related to genome expression and integrity such us transcription termination, the regulation of transcription-replication conflicts and the resolution of R-loops. SETX has been the focus of numerous studies since the discovery that mutations in its coding gene are the root cause of two different neurodegenerative diseases: Ataxia with Oculomotor Apraxia type 2 (AOA2) and a juvenile form of Amyotrophic Lateral Sclerosis (ALS4). A plethora of cellular phenotypes have been described as the result of SETX deficiency, yet the precise molecular function of SETX as well as the molecular pathways leading from SETX mutations to AOA2 and ALS4 pathologies have remained unclear. However, recent data have shed light onto the biochemical activities and biological roles of SETX, thus providing new clues to understand the molecular consequences of SETX mutation. In this review we summarize near two decades of scientific effort to elucidate SETX function, we discuss strengths and limitations of the approaches and models used thus far to investigate SETX-associated diseases and suggest new possible research avenues for the study of AOA2 and ALS4 pathogenesis. • SETX play roles in transcription termination and the regulation of gene expression. • SETX is crucial for R-loops control and the maintenance of genome stability. • SETX is an RNA/DNA helicase with evolutionary conserved biochemical activities. • Mutations in the SETX gene are linked to two distinct neurodegenerative diseases. [ABSTRACT FROM AUTHOR]

Telomeres and associated proteins safeguard chromosome ends to preserve genome stability. Destabilization of telomeres leads to fusion of telomeres and accumulation of DNA damage. Telomeres and retrotransposons exhibit reciprocal regulation of each other. Shortest telomeres can cause genome instability by epigenetic activation of retrotransposons. Telomeres and their associated proteins protect the ends of chromosomes to maintain genome stability. Telomeres undergo progressive shortening with each cell division in mammalian somatic cells without telomerase, resulting in genome instability. When telomeres reach a critically short length or are recognized as a damage signal, cells enter a state of senescence, followed by cell cycle arrest, programmed cell death, or immortalization. This review provides an overview of recent advances in the intricate relationship between telomeres and genome instability. Alongside well-established mechanisms such as chromosomal fusion and telomere fusion, we will delve into the perspective on genome stability by examining the role of retrotransposons. Retrotransposons represent an emerging pathway to regulate genome stability through their interactions with telomeres. [ABSTRACT FROM AUTHOR]

The Zinc finger and SCAN domain containing 4 (ZSCAN4) protein, expressed transiently in pluripotent stem cells, gametes, and early embryos, extends telomeres, enhances genome stability, and improves karyotypes in mouse embryonic stem (mES) cells. To gain insights into the mechanism of ZSCAN4 function, we identified genome-wide binding sites of endogenous ZSCAN4 protein using ChIP-seq technology in mouse and human ES cells, where the expression of endogenous ZSCAN4 was induced by treating cells with retinoic acids or by overexpressing DUX4. We revealed that both mouse and human ZSCAN4 bind to the TGCACAC motif located in CA/TG microsatellite repeats, which are known to form unstable left-handed duplexes called Z-DNA that can induce double-strand DNA breaks and mutations. These ZSCAN4 binding sites are mostly located in intergenic and intronic regions of the genomes. By generating ZSCAN4 knockout in human ES cells, we showed that ZSCAN4 does not seem to be involved in transcriptional regulation. We also found that ectopic expression of mouse ZSCAN4 enhances the suppression of chromatin at ZSCAN4-binding sites. These results together suggest that some of the ZSCAN4 functions are mediated by binding to the error-prone regions in mouse and human genomes. [ABSTRACT FROM AUTHOR]

Genome damage and transcription are intimately linked. Tens to hundreds of thousands of DNA lesions arise in each cell each day, many of which can directly or indirectly impede transcription. Conversely, the process of gene expression is itself a source of endogenous DNA lesions as a result of the susceptibility of single-stranded DNA to damage, conflicts with the DNA replication machinery, and engagement by cells of topoisomerases and base excision repair enzymes to regulate the initiation and progression of gene transcription. Although such processes are tightly regulated and normally accurate, on occasion, they can become abortive and leave behind DNA breaks that can drive genome rearrangements, instability, or cell death. DNA damage and transcription are intimately linked, with DNA lesions that impede transcription arising continuously and transcription itself being a source of DNA damage. Here, we review the complex interplay between DNA damage and transcription and the potential contribution of this interplay to human disease. [ABSTRACT FROM AUTHOR]

Genome stability is key for healthy cells in healthy organisms, and deregulated maintenance of genome integrity is a hallmark of aging and of age-associated diseases including cancer and neurodegeneration. To maintain a stable genome, genome surveillance and repair pathways are closely intertwined with cell cycle regulation and with DNA transactions that occur during transcription and DNA replication. Coordination of these processes across different time and length scales involves dynamic changes of chromatin topology, clustering of fragile genomic regions and repair factors into nuclear repair centers, mobilization of the nuclear cytoskeleton, and activation of cell cycle checkpoints. Here, we provide a general overview of cell cycle regulation and of the processes involved in genome duplication in human cells, followed by an introduction to replication stress and to the cellular responses elicited by perturbed DNA synthesis. We discuss fragile genomic regions that experience high levels of replication stress, with a particular focus on telomere fragility caused by replication stress at the ends of linear chromosomes. Using alternative lengthening of telomeres (ALT) in cancer cells and ALT-associated PML bodies (APBs) as examples of replication stress-associated clustered DNA damage, we discuss compartmentalization of DNA repair reactions and the role of protein properties implicated in phase separation. Finally, we highlight emerging connections between DNA repair and mechanobiology and discuss how biomolecular condensates, components of the nuclear cytoskeleton, and interfaces between membrane-bound organelles and membraneless macromolecular condensates may cooperate to coordinate genome maintenance in space and time. [ABSTRACT FROM AUTHOR]

Transcription-replication conflict is a major cause of replication stress that arises when replication forks collide with the transcription machinery. Replication fork stalling at sites of transcription compromises chromosome replication fidelity and can induce DNA damage with potentially deleterious consequences for genome stability and organismal health. The block to DNA replication by the transcription machinery is complex and can involve stalled or elongating RNA polymerases, promoter-bound transcription factor complexes, or DNA topology constraints. In addition, studies over the past two decades have identified co-transcriptional R-loops as a major source for impairment of DNA replication forks at active genes. However, how R-loops impede DNA replication at the molecular level is incompletely understood. Current evidence suggests that RNA:DNA hybrids, DNA secondary structures, stalled RNA polymerases, and condensed chromatin states associated with R-loops contribute to the of fork progression. Moreover, since both R-loops and replication forks are intrinsically asymmetric structures, the outcome of R-loop-replisome collisions is influenced by collision orientation. Collectively, the data suggest that the impact of R-loops on DNA replication is highly dependent on their specific structural composition. Here, we will summarize our current understanding of the molecular basis for R-loop-induced replication fork progression defects. [ABSTRACT FROM AUTHOR]

The whole genome sequence (WGS) of Bacillus coagulans BCP92 is reported along with its genomic analysis of probiotics and safety features. The identification of bacterial strain was carried out using the 16S rDNA sequencing method. Furthermore, gene-related probiotic features, safety assessment (by in vitro and in silico), and genome stability were also studied using the WGS analysis for the possible use of the bacterial strain as a probiotic. From the BLAST analysis, bacterial strain was identified as Bacillus (Heyndrickxia) coagulans. WGS analysis indicated that the genome consists of a 3 475 658 bp and a GC-content of 46.35%. Genome mining of BCP92 revealed that the strain is consist of coding sequences for d -lactate dehydrogenase and l -lactate dehydrogenases, 36 genes involved in fermentation activities, 29 stress-responsive as well as many adhesions related genes. The genome, also possessing genes, is encoded for the synthesis of novel circular bacteriocin. Using an in-silico approach for the bacterial genome study, it was possible to determine that the Bacillus (Heyndrickxia) coagulans strain BCP92 contains genes that are encoded for the probiotic abilities and did not harbour genes that are risk associated, thus confirming the strain's safety and suitability as a probiotic to be used for human application. [ABSTRACT FROM AUTHOR]

Telomeres protect the ends of linear eukaryotic chromosomes from being recognized as DNA double-strand breaks. Two major protein complexes are involved in the protection of telomeres: shelterin and CST. The dysfunction of these complexes can challenge the function of telomeres and lead to telomere fusions, breakage–fusion–bridge cycles, and cell death. Therefore, monitoring telomere fusions helps to understand telomeres biology. Telomere fusions are often analyzed by Fluorescent In Situ Hybridization (FISH) or PCR. Usually, both methods involve hybridization with a telomeric probe, which allows the detection of fusions containing telomeric sequences, but not of those lacking them. With the aim of detecting both types of fusion events, we have developed a nested PCR method to analyze telomere fusions in Arabidopsis thaliana. This method is simple, accurate, and does not require hybridization. We have used it to analyze telomere fusions in wild-type and mutant plants altered in CTC1, one of the three components of the Arabidopsis CST telomere capping complex. Our results show that null ctc1-2 mutant plants display fusions between all telomeric regions present in Arabidopsis chromosomes 1, 3 and 5, thus highlighting the widespread end-capping protection achieved by CTC1. [ABSTRACT FROM AUTHOR]

5-Hydroxymethfurural (5-HMF) is naturally found in a variety of foods and beverages and represents a main inhibitor in the lignocellulosic hydrolysates used for fermentation. This study investigated the impact of 5-HMF on the genomic stability and phenotypic plasticity of the yeast Saccharomyces cerevisiae. Using next-generation sequencing technology, we examined the genomic alterations of diploid S. cerevisiae isolates that were subcultured on a medium containing 1.2 g/L 5-HMF. We found that in 5-HMF-treated cells, the rates of chromosome aneuploidy, large deletions/duplications, and loss of heterozygosity were elevated compared with that in untreated cells. 5-HMF exposure had a mild impact on the rate of point mutations but altered the mutation spectrum. Contrary to what was observed in untreated cells, more monosomy than trisomy occurred in 5-HMF-treated cells. The aneuploidy mutant with monosomic chromosome IX was more resistant to 5-HMF than the diploid parent strain because of the enhanced activity of alcohol dehydrogenase. Finally, we found that overexpression of ADH6 and ZWF1 effectively stabilized the yeast genome under 5-HMF stress. Our findings not only elucidated the global effect of 5-HMF on the genomic integrity of yeast but also provided novel insights into how chromosomal instability drives the environmental adaptability of eukaryotic cells. [ABSTRACT FROM AUTHOR]

Mutations present a dichotomy in their implications for cellular processes. They primarily arise from DNA replication errors or damage repair processes induced by environmental challenges. Cumulative mutations underlie genetic variations and drive evolution, yet also contribute to degenerative diseases such as cancer and aging. The mutator phenotype elucidates the heightened mutation rates observed in malignant tumors. Evolutionary adaptation, analogous to bacterial and eukaryotic systems, manifests through mutator phenotypes during changing environmental conditions, highlighting the delicate balance between advantageous mutations and their potentially detrimental consequences. Leveraging the genetic tractability of Saccharomyces cerevisiae offers unique insights into mutator phenotypes and genome instability akin to human cancers. Innovative reporter assays in yeast model organisms enable the detection of diverse genome alterations, aiding a comprehensive analysis of mutator phenotypes. Despite significant advancements, our understanding of the intricate mechanisms governing spontaneous mutation rates and preserving genetic integrity remains incomplete. This review outlines various cellular pathways affecting mutation rates and explores the role of mutator genes and mutation-derived phenotypes, particularly prevalent in malignant tumor cells. An in-depth comprehension of mutator and antimutator activities in yeast and higher eukaryotes holds promise for effective cancer control strategies. [ABSTRACT FROM AUTHOR]

Abstract Synthetic lethality (SL), a genetic concept, has revolutionized the development of antitumor therapies by providing avenues to target previously “undruggable” targets with enhanced specificity for tumor cells over normal tissue. The principles of SL have expanded beyond genetic definitions to encompass biological functions, including genome stability, cell cycle regulation, cell death mechanisms, cellular metabolism, cell–cell interactions, and the tumor microenvironment (TME). Tumor cells with inactivated survival pathways are sensitive to therapeutic inhibition of compensatory mechanisms, while normal cells remain unaffected. Exploiting SL based on functional contexts has the potential to significantly improve cancer patient survival by reducing resistance to targeted therapies and enhancing antitumor efficacy when combined with other treatment modalities. This review explores the underlying mechanisms of synthetic lethality interactions (SLI) characterized by biological functions in individual cancer cells and the TME. We also provide a comprehensive summary of strategies for leveraging the dynamic nature of SLI to overcome therapeutic resistance. Additionally, we discuss various approaches and models for screening and designing potent SL agents tailored to the specific needs of cancer patients, as well as strategies for combining SL drugs in tumor treatment. This review offers valuable insights into harnessing SL as a promising avenue for precision cancer therapy.

Introduction: The R-loop is a naturally formed three-strand nucleic acid structure that recently has been reported to participate in multiple biological processes and helped answer some previously unexplained scientific questions. Meiosis process involves multiple chromatin-related events such as DNA double-stranded breaks (DSB) formation, repairing and transcriptional dynamics. Objectives: Explore the regulatory roles and physiological functions of R-loops in the mammalian meiosis process. Methods: In our study, using genome-wide S9.6 CUT & Tag seq, we first mapped the genomic distribution and dynamic changes of R-loop during the meiotic process in mice, from spermatogonia to secondary spermatocytes. And we further explore the role of R-loop in physiological conditions by constructing conditional knockout mice of Rnaseh1, which deleted the R-loop endonuclease before meiosis entry. Results: R-loop predominantly distributes at promoter-related regions and varies across different meiotic stages. By joint analysis with the corresponding transcriptome, we found that the R-loop was closely related to transcription during the meiotic process. The high frequency of promoter-related R-loop in meiotic cells is usually accompanied by high transcription activity, and we further verified this in the leptotene/zygotene to the pachytene transition process. Moreover, the lack of RNase H1 caused sterility in male mice with R-loop accumulation and abnormal DSB repair in spermatocytes. Further analysis showed that abnormal R-loop accumulation in the leptotene/zygotene stages influenced transcriptional regulation in the pachytene stage. Conclusion: The mutual regulation of the R-loop and transcription plays an essential role in spermatogenesis. And R-loop is also important for the normal repair process of DSB during meiosis.

Evolutionary selection ensures specificity and efficiency in dynamic metastable macromolecular machines that repair DNA damage without releasing toxic and mutagenic intermediates. Here we examine non-homologous end joining (NHEJ) as the primary conserved DNA double-strand break (DSB) repair process in human cells. NHEJ has exemplary key roles in networks determining the development, outcome of cancer treatments by DSB-inducing agents, generation of antibody and T-cell receptor diversity, and innate immune response for RNA viruses. We determine mechanistic insights into NHEJ structural biochemistry focusing upon advanced small angle X-ray scattering (SAXS) results combined with X-ray crystallography (MX) and cryo-electron microscopy (cryo-EM). SAXS coupled to atomic structures enables integrated structural biology for objective quantitative assessment of conformational ensembles and assemblies in solution, intra-molecular distances, structural similarity, functional disorder, conformational switching, and flexibility. Importantly, NHEJ complexes in solution undergo larger allosteric transitions than seen in their cryo-EM or MX structures. In the long-range synaptic complex, X-ray repair cross-complementing 4 (XRCC4) plus XRCC4-like-factor (XLF) form a flexible bridge and linchpin for DNA ends bound to KU heterodimer (Ku70/80) and DNA-PKcs (DNA-dependent protein kinase catalytic subunit). Upon binding two DNA ends, auto-phosphorylation opens DNA-PKcs dimer licensing NHEJ via concerted conformational transformations of XLF-XRCC4, XLF-Ku80, and LigIVBRCT -Ku70 interfaces. Integrated structures reveal multifunctional roles for disordered linkers and modular dynamic interfaces promoting DSB end processing and alignment into the short-range complex for ligation by LigIV. Integrated findings define dynamic assemblies fundamental to designing separation-of-function mutants and allosteric inhibitors targeting conformational transitions in multifunctional complexes.

Abstract Background The maintenance of genome stability in primordial germ cells (PGCs) is crucial for the faithful transmission of genetic information and the establishment of reproductive reserve. Numerous studies in recent decades have linked the Fanconi anemia (FA) pathway with fertility, particularly PGC development. However, the role of FAAP100, an essential component of the FA core complex, in germ cell development is unexplored. Results We find that FAAP100 plays an essential role in R-loop resolution and replication fork protection to counteract transcription-replication conflicts (TRCs) during mouse PGC proliferation. FAAP100 deletion leads to FA pathway inactivation, increases TRCs as well as cotranscriptional R-loops, and contributes to the collapse of replication forks and the generation of DNA damage. Then, the activated p53 signaling pathway triggers PGC proliferation defects, ultimately resulting in insufficient establishment of reproductive reserve in both sexes of mice. Conclusions Our findings suggest that FAAP100 is required for the resolution of TRCs in PGCs to safeguard their genome stability.

Sequences that form DNA secondary structures, such as G‐quadruplexes (G4s) and intercalated‐Motifs (iMs), are abundant in the human genome and play various physiological roles. However, they can also interfere with replication and threaten genome stability. Multiple lines of evidence suggest G4s inhibit replication, but the underlying mechanism remains unclear. Moreover, evidence of how iMs affect the replisome is lacking. Here, we reconstitute replication of physiologically derived structure‐forming sequences to find that a single G4 or iM arrest DNA replication. Direct single‐molecule structure detection within solid‐state nanopores reveals structures form as a consequence of replication. Combined genetic and biophysical characterisation establishes that structure stability and probability of structure formation are key determinants of replisome arrest. Mechanistically, replication arrest is caused by impaired synthesis, resulting in helicase‐polymerase uncoupling. Significantly, iMs also induce breakage of nascent DNA. Finally, stalled forks are only rescued by a specialised helicase, Pif1, but not Rrm3, Sgs1, Chl1 or Hrq1. Altogether, we provide a mechanism for quadruplex structure formation and resolution during replication and highlight G4s and iMs as endogenous sources of replication stress. Synopsis: By reconstituting eukaryotic DNA replication in vitro, this study addresses the effects of physiological quadruplex secondary structures on genome stability. G‐quadruplexes (G4s) and intercalated Motifs (iMs) are found to form during replication and thereby induce replisome stalling, leading to helicase‐polymerase uncoupling and nascent DNA breakage. A single physiological G4 or iM structure stalls the eukaryotic replisome by inhibiting leading strand synthesis.Helicase‐polymerase uncoupling occurs following replication stalling at G4s.iMs can induce breakage on nascent DNA.Stalled forks at G4s or iMs can be rescued by the accessory helicase Pif1. [ABSTRACT FROM AUTHOR]

Transcription elongation requires elaborate coordination between the transcriptional machinery and chromatin regulatory factors to successfully produce RNA while preserving the epigenetic landscape. Recent structural and genomic studies have highlighted that suppressor of Ty 6 (Spt6), a conserved histone chaperone and transcription elongation factor, sits at the crux of the transcription elongation process. Other recent studies have revealed that Spt6 also promotes DNA replication and genome integrity. Here, we review recent studies of Spt6 that have provided new insights into the mechanisms by which Spt6 controls transcription and have revealed the breadth of Spt6 functions in eukaryotic cells. Spt6 is a structural hub of the activated RNA polymerase II (RNAPII) elongation complex and forms critical contacts with RNAPII as well as other elongation factors, including DRB sensitivity-inducing factor (DSIF), polymerase-associated factor 1 complex (Paf1C), Iws1, and FACT. Spt6 directly interacts with all four core histones and nucleosomes and can assemble nucleosomes in vitro. Spt6 promotes processivity and elongation rate past nucleosomal barriers. Spt6 regulates nucleosome positioning and several histone post-translational modifications, thereby repressing intragenic transcription. Spt6 is necessary for genome stability, likely due to its roles in both transcription and DNA replication. [ABSTRACT FROM AUTHOR]

Exercise transiently impacts the expression, regulation, and activity of TERT/telomerase to maintain telomeres and protect the genome from insults. By protecting the telomeres (chromosome ends) and the genome, telomerase promotes cellular survival and prevents cellular senescence. By increasing cellular resiliency, via the actions of telomerase and TERT, exercise promotes healthy aging. [ABSTRACT FROM AUTHOR]