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Alternative end joining (alt-EJ) mechanisms, such as polymerase theta-mediated end joining, are increasingly recognized as important contributors to inaccurate double-strand break repair. We previously proposed an alt-EJ model whereby short DNA repeats near a double-strand break anneal to form secondary structures that prime limited DNA synthesis. The nascent DNA then pairs with microhomologous sequences on the other break end. This synthesis-dependent microhomology-mediated end joining (SD-MMEJ) explains many of the alt-EJ repair products recovered following I-SceI nuclease cutting in Drosophila. However, sequence-specific factors that influence SD-MMEJ repair remain to be fully characterized. Here, we expand the utility of the SD-MMEJ model through computational analysis of repair products at Cas9-induced double-strand breaks for 1100 different sequence contexts. We find evidence at single nucleotide resolution for sequence characteristics that drive successful SD-MMEJ repair. These include optimal primer repeat length, distance of repeats from the break, flexibility of DNA sequence between primer repeats, and positioning of microhomology templates relative to preferred primer repeats. In addition, we show that DNA polymerase theta is necessary for most SD-MMEJ repair at Cas9 breaks. The analysis described here includes a computational pipeline that can be utilized to characterize preferred mechanisms of alt-EJ repair in any sequence context.

Pericentromeric heterochromatin is highly enriched for repetitive sequences prone to aberrant recombination. Previous studies showed that homologous recombination (HR) repair is uniquely regulated in this domain to enable ‘safe’ repair while preventing aberrant recombination. InDrosophilacells, DNA double-strand breaks (DSBs) relocalize to the nuclear periphery through nuclear actin-driven directed motions before recruiting the strand invasion protein Rad51 and completing HR. End-joining (EJ) repair also occurs with high frequency in heterochromatin of fly tissues, but how different EJ pathways operate in heterochromatin remains uncharacterized. Here, we induce DSBs in single euchromatic and heterochromatic sites using the DR-whitereporter and I-SceI expression in spermatogonia. We detect higher frequency of HR repair in heterochromatic insertions, relative to euchromatin. Sequencing of repair outcomes reveals the use of distinct EJ pathways across different euchromatic and heterochromatic sites. Interestingly, synthesis-dependent michrohomology-mediated end joining (SD-MMEJ) appears differentially regulated in the two domains, with a preferential use of motifs close to the cut site in heterochromatin relative to euchromatin, resulting in smaller deletions. Together, these studies establish a new approach to study repair outcomes in fly tissues, and support the conclusion that heterochromatin uses more HR and less mutagenic EJ repair relative to euchromatin.

Deshpande et al. (2022) demonstrate that BRCA1, a tumor suppressor tasked with protecting the genome, is encoded by a gene that is intrinsically fragile.

Rif1 plays important roles in the repair of DNA double-strand breaks in multiple organisms. In mammals, RIF1 promotes non-homologous end joining and suppresses homologous recombination by interacting with 53BP1 to inhibit resection. In Saccharomyces cerevisiae, Rif1 directly binds DNA to inhibit resection and promote non-homologous end-joining. Yeast Rif1 can also facilitate long-range resection and promote single-strand annealing. Since it is not clear if Rif1 regulates resection-mediated pathway choice in other eukaryotes, we explored the role of Rif1 in double-strand break repair in Drosophila melanogaster. We found that rif1 mutants are not sensitive to ionizing radiation or hydroxyurea, demonstrating that it is not essential for the resolution of DNA damage in Drosophila. However, we show that rif1 null mutants are largely unable to repair a specific type of double-strand break that is induced upon the excision of a P-element transposon. Furthermore, assessment of repair pathway choice at I-SceI-induced breaks revealed Rif1 suppresses homologous recombination and promotes single-strand annealing. Collectively, our findings illustrate Drosophila Rif1 shares functions with both its yeast and mammalian counterparts and serves a unique role in repairing P-element-induced double-strand breaks.

Repair of DNA double-strand breaks by homologous recombination (HR) requires a carefully orchestrated sequence of events involving many proteins. One type of HR, synthesis-dependent strand annealing (SDSA), proceeds via the formation of a displacement loop (D-loop) when RAD51-coated single-stranded DNA invades a homologous template. The 3′ end of the single-stranded DNA is extended by DNA synthesis. In SDSA, the D-loop is then disassembled prior to strand annealing. While many helicases can unwind D-loops in vitro, how their action is choreographed in vivo remains to be determined. To clarify the roles of various DNA helicases during SDSA, we used a double-strand gap repair assay to study the outcomes of homologous recombination repair in Drosophila melanogaster lacking the BLM, HELQ, and FANCM helicases. We found that the absence of any of these three helicases impairs gap repair. In addition, flies lacking both BLM and HELQ or HELQ and FANCM had more severe SDSA defects than the corresponding single mutants. In the absence of BLM, a large percentage of repair events were accompanied by flanking deletions. Strikingly, these deletions were mostly abolished in the blm helq and blm fancm double mutants. Our results suggest that the BLM, HELQ, and FANCM helicases play distinct roles during SDSA, with HELQ and FANCM acting early to promote the formation of recombination intermediates that are then processed by BLM to prevent repair by deletion-prone mechanisms.

Traditional assessments in college biology classrooms, such as exams and lab reports, often have limited utility in promoting long-lasting understanding of course material and do not always engage students from all backgrounds. The inclusion of creative scientific writing assignments, especially those that require application of sophisticated course material, is an underutilized strategy in higher education. Here, we describe our use of student-generated poetry in two midlevel undergraduate biology classes. We have found that by encouraging students to write poems in response to carefully crafted prompts and having them assess the scientific accuracy of the poems, we can encourage them to identify misconceptions prior to exams, potentially resulting in deeper and longer-lasting understanding of course material. Furthermore, the inclusion of poetry empowers students who might not otherwise participate in class to contribute, resulting in a more inclusive classroom climate.

Sertraline hydrochloride is a commonly prescribed antidepressant medication that acts by amplifying serotonin signaling. Numerous studies have suggested that children of women taking sertraline during pregnancy have an increased risk of developmental defects. Resolving the degree of risk for human fetuses requires comprehensive knowledge of the pathways affected by this drug. We utilized a Drosophila melanogaster model system to assess the effects of sertraline throughout development. Ingestion of sertraline by females did not affect their fecundity or embryogenesis in their progeny. However, larvae that consumed sertraline experienced delayed developmental progression and reduced survival at all stages of development. Genetic experiments showed that these effects were mostly independent of aberrant extracellular serotonin levels. Using an ex vivo imaginal disc culture system, we showed that mitotically active sertraline-treated tissues accumulate DNA double-strand breaks and undergo apoptosis at increased frequencies. Remarkably, the sertraline-induced genotoxicity was partially rescued by co-incubation with ascorbic acid, suggesting that sertraline induces oxidative DNA damage. These findings may have implications for the biomedicine of sertraline-induced birth defects.

PIF1 is a 5′ to 3′ DNA helicase that can unwind double-stranded DNA and disrupt nucleic acid-protein complexes. In Saccharomyces cerevisiae, Pif1 plays important roles in mitochondrial and nuclear genome maintenance, telomere length regulation, unwinding of G-quadruplex structures, and DNA synthesis during break-induced replication. Some, but not all, of these functions are shared with other eukaryotes. To gain insight into the evolutionarily conserved functions of PIF1, we created pif1 null mutants in Drosophila melanogaster and assessed their phenotypes throughout development. We found that pif1 mutant larvae exposed to high concentrations of hydroxyurea, but not other DNA damaging agents, experience reduced survival to adulthood. Embryos lacking PIF1 fail to segregate their chromosomes efficiently during early nuclear divisions, consistent with a defect in DNA replication. Furthermore, loss of the BRCA2 protein, which is required for stabilization of stalled replication forks in metazoans, causes synthetic lethality in third instar larvae lacking either PIF1 or the polymerase delta subunit POL32. Interestingly, pif1 mutants have a reduced ability to synthesize DNA during repair of a double-stranded gap, but only in the absence of POL32. Together, these results support a model in which Drosophila PIF1 functions with POL32 during times of replication stress but acts independently of POL32 to promote synthesis during double-strand gap repair.

Corticosterone does not change in consistent ways across species and contexts, making it challenging to use as an indicator of chronic stress. We assessed DNA damage as a potential metric that could be a more integrative stress measurement with direct links to health. We captured free-living house sparrows, took an immediate blood sample, and transferred them to the laboratory, exposing them to the chronic stress of captivity. Biweekly blood and weight samples were then taken for 4 weeks. We immediately assessed DNA damage in red blood cells using the comet assay and later quantified corticosterone. Uric acid was analyzed in a separate group of birds. We found that birds initially lost, but began to regain weight over the course of captivity. DNA damage peaked within the first 10 days of captivity, and mostly remained elevated. However, the cellular distribution of damage changed considerably over time; most cells showed low levels of damage early, a bimodal distribution of high and low DNA damage during the peak of damage, and a wide unimodal distribution of damage at the end of the 4 weeks. Furthermore, corticosterone increased and remained elevated and uric acid decreased and remained depleted over the same period. Although both a molecular (DNA damage) and an endocrine (corticosterone) marker showed similar response profiles over the 4 weeks, they were not correlated, suggesting they reflect different aspects of the underlying physiology. These data provide convincing preliminary evidence that DNA damage has potential to be an additional indicator of chronic stress.

Although corticosterone (Cort) has been the predominant metric used to assess acute stress in birds, it does not always accurately reflect how an animal copes with a stressor. Downstream measurements may be more reliable. In the current study, we tested the hypothesis that acute increases in DNA damage could be used to assess stressor exposure. Studies have shown DNA damage increases in response to stress-related hormones in vitro; however, this has not yet been thoroughly applied in wild animals. We exposed house sparrows (Passer domesticus) to a 30- or 120-min restraint stressor and took blood samples at 0, 30, 60, and 120 min to measure Cort, DNA damage, and uric acid. Both treatments increased DNA damage and Cort, and decreased uric acid. It thus appears that DNA damage can reflect acute stressor exposure. To improve the usability of DNA damage as a metric for stress, we also tested the impacts of sample storage on DNA damage. Leaving red blood cells on ice for up to 24 hr, only slightly influenced DNA damage. Freezing blood samples for 1-4 weeks substantially increased DNA damage. These findings emphasize the importance of reducing variation between samples by assaying them together whenever possible. Overall, these results indicate that assessing DNA damage is a valid method of assessing acute stressor exposure that is suitable for both laboratory- and field-based studies; however, additional research is needed on the molecular dynamics of nucleated red blood cells, including whether and how their DNA is repaired.

DNA synthesis during replication or repair is a fundamental cellular process that is catalyzed by a set of evolutionary conserved polymerases. Despite a large body of research, the DNA polymerases of Drosophila melanogaster have not yet been systematically reviewed, leading to inconsistencies in their nomenclature, shortcomings in their functional (Gene Ontology, GO) annotations and an under-appreciation of the extent of their characterization. Here, we describe the complete set of DNA polymerases in D. melanogaster, applying nomenclature already in widespread use in other species, and improving their functional annotation. A total of 19 genes encode the proteins comprising three replicative polymerases (alpha-primase, delta, epsilon), five translesion/repair polymerases (zeta, eta, iota, Rev1, theta) and the mitochondrial polymerase (gamma). We also provide an overview of the biochemical and genetic characterization of these factors in D. melanogaster. This work, together with the incorporation of the improved nomenclature and GO annotation into key biological databases, including FlyBase and UniProtKB, will greatly facilitate access to information about these important proteins.

PIF1 is a 5′ to 3′ DNA helicase that can unwind double-stranded DNA and disrupt nucleic acid-protein complexes. In Saccharomyces cerevisiae, Pif1 plays important roles in mitochondrial and nuclear genome maintenance, telomere length regulation, unwinding of G-quadruplex structures, and DNA synthesis during break-induced replication. Some, but not all, of these functions are shared with other eukaryotes. To gain insight into the evolutionarily conserved functions of PIF1, we created pif1 null mutants in Drosophila melanogaster and assessed their phenotypes throughout development. We found that pif1 mutant larvae exposed to high concentrations of hydroxyurea, but not other DNA damaging agents, experience reduced survival to adulthood. Embryos lacking PIF1 fail to segregate their chromosomes efficiently during early nuclear divisions, consistent with a defect in DNA replication. Furthermore, loss of the BRCA2 protein, which is required for stabilization of stalled replication forks in metazoans, causes synthetic lethality in third instar larvae lacking either PIF1 or the polymerase delta subunit POL32. Interestingly, pif1 mutants have a reduced ability to synthesize DNA during repair of a double-stranded gap, but only in the absence of POL32. Together, these results support a model in which Drosophila PIF1 functions with POL32 during times of replication stress but acts independently of POL32 to promote synthesis during double-strand gap repair.

In this chapter, we describe a method for the recovery and analysis of alternative end-joining (alt-EJ) DNA double-strand break repair junctions following I-SceI cutting in Drosophila melanogaster embryos. Alt-EJ can be defined as a set of Ku70/80 and DNA ligase 4-independent end-joining processes that are typically mutagenic, producing deletions, insertions, and chromosomal rearrangements more frequently than higher-fidelity repair pathways such as classical nonhomologous end joining or homologous recombination. Alt-EJ has been observed to be upregulated in HR-deficient tumors and is essential for the survival and proliferation of these cells. Alt-EJ shares many initial processing steps with homologous recombination, specifically end resection; therefore, studying alt-EJ repair junctions can provide useful insight into aborted HR repair. Here, we describe the injection of plasmid constructs with specific cut sites into Drosophila embryos and the subsequent recovery of alt-EJ repair products. We also describe different analytical approaches using this system and how amplicon sequencing can be used to provide mechanistic information about alt-EJ.

In this chapter, we describe a method for the recovery and analysis of alternative end-joining (alt-EJ) DNA double-strand break repair junctions following I-SceI cutting in Drosophila melanogaster embryos. Alt-EJ can be defined as a set of Ku70/80 and DNA ligase 4-independent end-joining processes that are typically mutagenic, producing deletions, insertions, and chromosomal rearrangements more frequently than higher-fidelity repair pathways such as classical nonhomologous end joining or homologous recombination. Alt-EJ has been observed to be upregulated in HR-deficient tumors and is essential for the survival and proliferation of these cells. Alt-EJ shares many initial processing steps with homologous recombination, specifically end resection; therefore, studying alt-EJ repair junctions can provide useful insight into aborted HR repair. Here, we describe the injection of plasmid constructs with specific cut sites into Drosophila embryos and the subsequent recovery of alt-EJ repair products. We also describe different analytical approaches using this system and how amplicon sequencing can be used to provide mechanistic information about alt-EJ.

Preserving the integrity of the DNA double helix is crucial for the maintenance of genomic stability. Therefore, DNA double-strand breaks represent a serious threat to cells. In this review, we describe the two major strategies used to repair double strand breaks: non-homologous end joining and homologous recombination, emphasizing the mutagenic aspects of each. We focus on emerging evidence that homologous recombination, long thought to be an error-free repair process, can in fact be highly mutagenic, particularly in contexts requiring large amounts of DNA synthesis. Recent investigations have begun to illuminate the molecular mechanisms by which error-prone double-strand break repair can create major genomic changes, such as translocations and complex chromosome rearrangements. We highlight these studies and discuss proposed models that may explain some of the more extreme genetic changes observed in human cancers and congenital disorders.

In Drosophila melanogaster, DNA double-strand breaks (DSBs) created by exposure to gamma or X-ray radiation can be quantified by immunofluorescent detection of phosphorylated histone H2Av (γ-H2Av) foci in imaginal disc tissues. This technique has been less useful for studying DSBs in imaginal discs exposed to DSB-inducing chemicals, since standard protocols require raising larvae in food treated with liquid chemical suspensions. These protocols typically take 3-4 days to complete and result in heterogeneous responses that do not provide information about the kinetics of DSB formation and repair. Here, we describe a novel and rapid method to quantify DSBs in imaginal discs cultured ex vivo with methyl methanesulfonate (MMS) or other DSB-inducing chemicals. The described method requires less than 24 h and provides precise control over MMS concentration and exposure time, enabling reproducible detection of transient DSBs. Furthermore, this technique can be used for nearly any chemical treatment and can be modified and adapted for several different experimental setups and downstream molecular analyses.

In Drosophila melanogaster, DNA double-strand breaks (DSBs) created by exposure to gamma or X-ray radiation can be quantified by immunofluorescent detection of phosphorylated histone H2Av (γ-H2Av) foci in imaginal disc tissues. This technique has been less useful for studying DSBs in imaginal discs exposed to DSB-inducing chemicals, since standard protocols require raising larvae in food treated with liquid chemical suspensions. These protocols typically take 3-4 days to complete and result in heterogeneous responses that do not provide information about the kinetics of DSB formation and repair. Here, we describe a novel and rapid method to quantify DSBs in imaginal discs cultured ex vivo with methyl methanesulfonate (MMS) or other DSB-inducing chemicals. The described method requires less than 24 h and provides precise control over MMS concentration and exposure time, enabling reproducible detection of transient DSBs. Furthermore, this technique can be used for nearly any chemical treatment and can be modified and adapted for several different experimental setups and downstream molecular analyses.

Members of the RecQ family of helicases are known for their roles in DNA repair, replication, and recombination. Mutations in the human RecQ helicases, WRN and BLM, cause Werner and Bloom syndromes, which are diseases characterized by genome instability and an increased risk of cancer. While WRN contains both a helicase and an exonuclease domain, the Drosophila melanogaster homolog, WRNexo, contains only the exonuclease domain. Therefore the Drosophila model system provides a unique opportunity to study the exonuclease functions of WRN separate from the helicase. We created a null allele of WRNexo via imprecise P-element excision. The null WRNexo mutants are not sensitive to double-strand break-inducing reagents, suggesting that the exonuclease does not play a key role in homologous recombination-mediated repair of DSBs. However, WRNexo mutant embryos have a reduced hatching frequency and larvae are sensitive to the replication fork-stalling reagent, hydroxyurea (HU), suggesting that WRNexo is important in responding to replication stress. The role of WRNexo in the HU-induced stress response is independent of Rad51. Interestingly, the hatching defect and HU sensitivity of WRNexo mutants do not occur in flies containing an exonuclease-dead copy of WRNexo, suggesting that the role of WRNexo in replication is independent of exonuclease activity. Additionally, WRNexo and Blm mutants exhibit similar sensitivity to HU and synthetic lethality in combination with mutations in structure-selective endonucleases. We propose that WRNexo and BLM interact to promote fork reversal following replication fork stalling and in their absence regressed forks are restarted through a Rad51-mediated process.

Werner syndrome (WS) is an autosomal recessive progeroid disease characterized by patients' early onset of aging, increased risk of cancer and other age-related pathologies. WS is caused by mutations in WRN, a RecQ helicase that has essential roles responding to DNA damage and preventing genomic instability. While human WRN has both an exonuclease and helicase domain, Drosophila WRNexo has high genetic and functional homology to only the exonuclease domain of WRN. Like WRN-deficient human cells, Drosophila WRNexo null mutants (WRNexoΔ) are sensitive to replication stress, demonstrating mechanistic similarities between these two models. Compared to age-matched wild-type controls, WRNexoΔ flies exhibit increased physiological signs of aging, such as shorter lifespans, higher tumor incidence, muscle degeneration, reduced climbing ability, altered behavior, and reduced locomotor activity. Interestingly, these effects are more pronounced in females suggesting sex-specific differences in the role of WRNexo in aging. This and future mechanistic studies will contribute to our knowledge in linking faulty DNA repair mechanisms with the process of aging.

The stress response represents an animal’s attempt to cope with a noxious stimulus through a rapid release of corticosterone or cortisol (CORT) into the bloodstream, resulting in a suite of physiological and behavioral changes. These changes are mediated in large part through CORT’s binding to two different intracellular receptors, the high-affinity mineralocorticoid receptor (MR) and the lower-affinity glucocorticoid receptor (GR). We tested the hypothesis that GR and MR mRNA expression would correlate with functional protein expression in neuronal tissue of wild-caught house sparrows (Passer domesticus). To test this hypothesis, we performed a parallel procedure in which protein concentrations were quantified in one half of house sparrow brains (n = 16) using radioligand binding assays, and mRNA levels were quantified in the other brain half using reverse-transcriptase quantitative PCR (RT-qPCR). Two reference genes, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and TATA-box binding protein (TBP), were used for relative quantification of GR and MR mRNA. Quantifications showed that these two reference genes were not correlated with each other. Furthermore, there was no correlation between mRNA and protein levels for GR or MR using either reference gene, suggesting that regulation of mRNA and protein levels for MR and GR is not tightly linked. This study provides insight into the importance of regulatory steps between mRNA expression and the creation and stability of a functional protein. The overall conclusion is that mRNA expression cannot be used as a proxy for GR or MR binding in house sparrows.

Many chemotherapeutic agents selectively target rapidly dividing cells, including cancer cells, by causing DNA damage that leads to genome instability and cell death. We used Drosophila melanogaster to study how mutations in key DNA repair genes affect an organism’s response to chemotherapeutic drugs. In this study, we focused on camptothecin and its derivatives, topotecan and irinotecan, which are type I topoisomerase inhibitors that create DNA double-strand breaks in rapidly dividing cells. Here, we describe two polymorphisms in Drosophila Cyp6d2 that result in extreme sensitivity to camptothecin but not topotecan or irinotecan. We confirmed that the sensitivity was due to mutations in Cyp6d2 by rescuing the defect with a wild-type copy of Cyp6d2. In addition, we showed that combining a cyp6d2 mutation with mutations in Drosophila brca2 results in extreme sensitivity to camptothecin. Given the frequency of the Cyp6d2 polymorphisms in publcly available Drosophila stocks, our study demonstrates the need for caution when interpreting results from drug sensitivity screens in Drosophila and other model organisms. Furthermore, our findings illustrate how genetic background effects can be important when determining the efficacy of chemotherapeutic agents in various DNA repair mutants.

Homologous recombination (HR) is a central process to ensure genomic stability in somatic cells and during meiosis. HR-associated DNA synthesis determines in large part the fidelity of the process. A number of recent studies have demonstrated that DNA synthesis during HR is conservative, less processive, and more mutagenic than replicative DNA synthesis. In this review, we describe mechanistic features of DNA synthesis during different types of HR-mediated DNA repair, including synthesis-dependent strand annealing, break-induced replication, and meiotic recombination. We highlight recent findings from diverse eukaryotic organisms, including humans, that suggest both replicative and translesion DNA polymerases are involved in HR-associated DNA synthesis. Our focus is to integrate the emerging literature about DNA polymerase involvement during HR with the unique aspects of these repair mechanisms, including mutagenesis and template switching.

Rereplication generates double-strand breaks (DSBs) at sites of fork collisions and causes genomic damage, including repeat instability and chromosomal aberrations. However, the primary mechanism used to repair rereplication DSBs varies across different experimental systems. In Drosophila follicle cells, developmentally regulated rereplication is used to amplify six genomic regions, two of which contain genes encoding eggshell proteins. We have exploited this system to test the roles of several DSB repair pathways during rereplication, using fork progression as a readout for DSB repair efficiency. Here we show that a null mutation in the microhomology-mediated end-joining (MMEJ) component, polymerase θ/mutagen-sensitive 308 (mus308), exhibits a sporadic thin eggshell phenotype and reduced chorion gene expression. Unlike other thin eggshell mutants, mus308 displays normal origin firing but reduced fork progression at two regions of rereplication. We also find that MMEJ compensates for loss of nonhomologous end joining to repair rereplication DSBs in a site-specific manner. Conversely, we show that fork progression is enhanced in the absence of both Drosophila Rad51 homologs, spindle-A and spindle-B, revealing homologous recombination is active and actually impairs fork movement during follicle cell rereplication. These results demonstrate that several DSB repair pathways are used during rereplication in the follicle cells and their contribution to productive fork progression is influenced by genomic position and repair pathway competition. Furthermore, our findings illustrate that specific rereplication DSB repair pathways can have major effects on cellular physiology, dependent upon genomic context.

Over the past two decades, a large collection of transposable elements inserted at various locations in the Drosophila melanogaster genome has been assembled. These transposons are frequently utilized in imprecise excision screens to generate deletions in genes of interest. In general, these screens involve genetic manipulations to combine a non-autonomous transposon and the appropriate transposase in individual male or female flies. DNA double-strand breaks are created via transposase action in both somatic and germline cells of these individuals and inaccurate repair events are recovered in the progeny. Because deletion-prone repair of transposon-induced double-strand breaks is rare, these screens generally require a significant investment of time and resources. We recently reported that conducting imprecise excision screens in mus309 mutant flies, which lack the Drosophila ortholog of the Bloom Syndrome helicase, results in an increase in both the number and size of deletions recovered. Here, we provide additional information for Drosophila researchers wishing to utilize this technique. In addition, we discuss how the general principle behind this technique can be applied in other contexts where double-strand breaks are being generated for the purpose of genome modification.

DNA polymerase theta (Pol θ) is an error-prone A-family polymerase that is highly conserved among multicellular eukaryotes and plays multiple roles in DNA repair and the regulation of genome integrity. Studies conducted in several model organisms have shown that Pol θ can be utilized during DNA interstrand crosslink repair and during alternative end-joining repair of double-strand breaks. Recent genetic and biochemical studies have begun to elucidate the unique structural features of Pol θ that promote alternative end-joining repair. Importantly, Pol θ-dependent end joining appears to be important for overall genome stability, as it affects chromosome translocation formation in murine and human cell lines. Pol θ has also been suggested to act as a modifier of replication timing in human cells, though the mechanism of action remains unknown. Pol θ is highly upregulated in a number of human cancer types, which could indicate that mutagenic Pol θ-dependent end joining is used during cancer cell proliferation. Here, we review the various roles of Pol θ across species and discuss how these roles may be relevant to cancer therapy.

DNA double-strand breaks (DSBs), a major source of genome instability, are often repaired through homologous recombination pathways. Models for these pathways have been proposed, but the precise mechanisms and the rules governing their use remain unclear. In Drosophila, the synthesis-dependent strand annealing (SDSA) model can explain most DSB repair. To investigate SDSA, we induced DSBs by excision of a P element from the male X chromosome, which produces a 14-kb gap relative to the sister chromatid. In wild-type males, repair synthesis tracts are usually long, resulting in frequent restoration of the P element. However, repair synthesis is often incomplete, resulting in internally deleted P elements. We examined the effects of mutations in spn-A, which encodes the Drosophila Rad51 ortholog. As expected, there is little or no repair synthesis in homozygous spn-A mutants after P excision. However, heterozygosity for spn-A mutations also resulted in dramatic reductions in the lengths of repair synthesis tracts. These findings support a model in which repair DNA synthesis is not highly processive. We discuss a model wherein repair of a double-strand gap requires multiple cycles of strand invasion, synthesis, and dissociation of the nascent strand. After dissociation, the nascent strand may anneal to a complementary single strand, reinvade a template to be extended by additional synthesis, or undergo end joining. This model can explain aborted SDSA repair events and the prevalence of internally deleted transposable elements in genomes.

We designed an interrupted case study to teach aerobic cellular respiration to major and nonmajor biology students. The case is based loosely on a real-life incident of rotenone poisoning. It places students in the role of a coroner who must determine the cause of death of the victim. The case is presented to the students in four parts. Each part is followed by discussion questions that the students answer in small groups prior to a classwide discussion. Successive parts of the case provide additional clues to the mystery and help the students focus on the physiological processes involved in aerobic respiration. Students learn the information required to solve the mystery by reading the course textbook prior to class, listening to short lectures interspersed throughout the case, and discussing the case in small groups. The case ends with small group discussions in which the students are given the names and specific molecular targets of other poisons of aerobic respiration and asked to determine which process (i.e., glycolysis, citric acid cycle, or the electron transport chain) the toxin disrupts.

Double strand breaks (DSBs) and interstrand crosslinks (ICLs) are toxic DNA lesions that can be repaired through multiple pathways, some of which involve shared proteins. One of these proteins, DNA Polymerase θ (Pol θ), coordinates a mutagenic DSB repair pathway named microhomology-mediated end joining (MMEJ) and is also a critical component for bypass or repair of ICLs in several organisms. Pol θ contains both polymerase and helicase-like domains that are tethered by an unstructured central region. While the role of the polymerase domain in promoting MMEJ has been studied extensively both in vitro and in vivo, a function for the helicase-like domain, which possesses DNA-dependent ATPase activity, remains unclear. Here, we utilize genetic and biochemical analyses to examine the roles of the helicase-like and polymerase domains of Drosophila Pol θ. We demonstrate an absolute requirement for both polymerase and ATPase activities during ICL repair in vivo. However, similar to mammalian systems, polymerase activity, but not ATPase activity, is required for ionizing radiation-induced DSB repair. Using a site-specific break repair assay, we show that overall end-joining efficiency is not affected in ATPase-dead mutants, but there is a significant decrease in templated insertion events. In vitro, Pol θ can efficiently bypass a model unhooked nitrogen mustard crosslink and promote DNA synthesis following microhomology annealing, although ATPase activity is not required for these functions. Together, our data illustrate the functional importance of the helicase-like domain of Pol θ and suggest that its tethering to the polymerase domain is important for its multiple functions in DNA repair and damage tolerance., Author summary Error-prone DNA Polymerase θ (Pol θ) plays a conserved role in a mutagenic DNA double-strand break repair mechanism called microhomology-mediated end joining (MMEJ). In many organisms, it also participates in a process crucial to the removal/repair of DNA interstrand crosslinks. The exact mechanism by which Pol θ promotes these processes is unclear, but a clue may lie in its dual-domain structure. While the role of its polymerase domain has been well-studied, the function of its helicase-like domain remains an open question. Here we report an absolute requirement for ATPase activity of the helicase-like domain during interstrand crosslink repair in Drosophila melanogaster. We also find that although end joining frequency does not decrease in ATPase-dead mutants, ATPase activity is critical for generating templated insertions. Using purified Pol θ protein, we show that it can bypass synthetic substrates mimicking interstrand crosslink intermediates and can promote MMEJ-like reactions with partial double-stranded and single-stranded DNA. Together, these data demonstrate a novel function for the helicase-like domain of Pol θ in both interstrand crosslink repair and MMEJ and provide insight into why the dual-domain structure has been conserved throughout evolution.

Werner syndrome (WS) is marked by early onset of features resembling aging, and is caused by loss of the RecQ family DNA helicase WRN. Precisely how loss of WRN leads to the phenotypes of WS is unknown. Cultured WS fibroblasts shorten their telomeres at an increased rate per population doubling and the premature senescence this loss induces can be bypassed by telomerase. Here we show that WRN co-localizes with telomeric factors in telomerase-independent immortalized human cells, and further that the budding yeast RecQ family helicase Sgs1p influences telomere metabolism in yeast cells lacking telomerase. Telomerase-deficient sgs1 mutants show increased rates of growth arrest in the G2/M phase of the cell cycle as telomeres shorten. In addition, telomerase-deficient sgs1 mutants have a defect in their ability to generate survivors of senescence that amplify telomeric TG1–3 repeats, and SGS1 functions in parallel with the recombination gene RAD51 to generate survivors. Our findings indicate that Sgs1p and WRN function in telomere maintenance, and suggest that telomere defects contribute to the pathogenesis of WS and perhaps other RecQ helicase diseases.

Silent information regulators, or Sir proteins, play distinct roles in chromatin-mediated transcriptional control at the silent mating-type loci, telomeres, and within the rDNA repeats of Saccharomyces cerevisiae. An unusual collection of sir3 mutant alleles was identified in a genetic screen for enhancers of the sir1 mutant mating-defective phenotype. These sir3-eso mutants, like the sir1 mutant, exhibit little or no mating defects alone, but the sir1 sir3-eso double mutants are essentially nonmating. All of the sir3-eso mutants are defective in telomeric silencing. In some mutants, this phenotype is suppressed by tethering Sir1p to telomeres; other mutants are dominant for mating and telomeric silencing defects. Additionally, several sir3-eso mutants are nonmating in combination with the nat1 N-terminal acetyltransferase mutant. The temperature-sensitive allele sir3-8 has an eso phenotype at permissive temperature, yet acts as a null allele at restrictive temperature due to loss of sir3-8 protein. Sequence analysis showed that eight of the nine sir3-eso alleles have mutations within the N-terminal region that is highly similar to the DNA replication initiation protein Orc1p. Together, these data reveal modular domains for Sir3p and further define its function in silencing chromatin.

The SIR genes are determinants of life span in yeast mother cells. Here we show that life span regulation by the Sir proteins is independent of their role in nonhomologous end joining. The short life span of a sir3 or sir4 mutant is due to the simultaneous expression of a and alpha mating-type information, which indirectly causes an increase in rDNA recombination and likely increases the production of extrachromosomal rDNA circles. The short life span of a sir2 mutant also reveals a direct failure to repress recombination generated by the Fob1p-mediated replication block in the rDNA. Sir2p is a limiting component in promoting yeast longevity, and increasing the gene dosage extends the life span in wild-type cells. A possible role of the conserved SIR2 in mammalian aging is discussed.

Many chemotherapeutic agents selectively target rapidly dividing cells, including cancer cells, by causing DNA damage that leads to genome instability and cell death. We used Drosophila melanogaster to study how mutations in key DNA repair genes affect an organism’s response to chemotherapeutic drugs. In this study, we focused on camptothecin and its derivatives, topotecan and irinotecan, which are type I topoisomerase inhibitors that create DNA double-strand breaks in rapidly dividing cells. Here, we describe two polymorphisms in Drosophila Cyp6d2 that result in extreme sensitivity to camptothecin but not topotecan or irinotecan. We confirmed that the sensitivity was due to mutations in Cyp6d2 by rescuing the defect with a wild-type copy of Cyp6d2. In addition, we showed that combining a cyp6d2 mutation with mutations in Drosophila brca2 results in extreme sensitivity to camptothecin. Given the frequency of the Cyp6d2 polymorphisms in publcly available Drosophila stocks, our study demonstrates the need for caution when interpreting results from drug sensitivity screens in Drosophila and other model organisms. Furthermore, our findings illustrate how genetic background effects can be important when determining the efficacy of chemotherapeutic agents in various DNA repair mutants.

Bloom syndrome, characterized by a predisposition to cancer, is caused by mutation of the RecQ DNA helicase gene BLM . The precise function of BLM remains unclear. Previous research suggested that Drosophila BLM functions in the repair of DNA double-strand breaks. Most double-strand breaks in flies are repaired by homologous recombination through the synthesis-dependent strand-annealing pathway. Here, we demonstrate that Drosophila BLM mutants are severely impaired in their ability to carry out repair DNA synthesis during synthesis-dependent strand annealing. Consequently, repair in the mutants is completed by error-prone pathways that create large deletions. These results suggest a model in which BLM maintains genomic stability by promoting efficient repair DNA synthesis and thereby prevents double-strand break repair by less precise pathways.

Background The BLM DNA helicase plays a vital role in maintaining genome stability. Mutations in BLM cause Bloom syndrome, a rare disorder associated with cancer predisposition and premature aging. Humans and mice with blm mutations have increased frequencies of spontaneous mutagenesis, but the molecular basis of this increase is not well understood. In addition, the effect of aging on spontaneous mutagenesis in blm mutants has not been characterized. To address this, we used a lacZ reporter system in wild-type and several mutant strains of Drosophila melanogaster to analyze mechanisms of mutagenesis throughout their lifespan. Results Our data show that Drosophila lacking BLM have an elevated frequency of spontaneous genome rearrangements that increases with age. Although in normal flies most genome rearrangements occur through DNA ligase 4-dependent classical end joining, most rearrangements that accumulate during aging in blm mutants do not require DNA ligase 4, suggesting the influence of an alternative end-joining mechanism. Adult blm mutants also display reduced lifespan and ligase 4-independent enhanced tumorigenesis in mitotically active tissues. Conclusions These results suggest that Drosophila BLM suppresses error-prone alternative end-joining repair of DNA double-strand breaks that can result in genome instability and tumor formation during aging. In addition, since loss of BLM significantly affects lifespan and tumorigenesis, the data provide a link between error-prone end joining, genome rearrangements, and tumor formation in a model metazoan.

This study investigated student learning outcomes using a case-based approach focused on cellular respiration. Students who used the case study, relative to students who did not use the case study, exhibited a significantly greater learning gain, and demonstrated use of higher-order thinking skills. Preliminary data indicate that after engaging with the case study, students were more likely to answer a question addressing misconceptions about cellular respiration correctly when compared with students who did not use the case study. More rigorous testing is needed to fully elucidate whether case-based learning can effectively clarify student misconceptions related to biological processes.

Ku or DNA ligase 4-independent alternative end joining (alt-EJ) repair of DNA double-strand breaks (DSBs) frequently correlates with increased junctional microhomology. However, alt-EJ also produces junctions without microhomology (apparent blunt joins), and the exact role of microhomology in both alt-EJ and classical non-homologous end joining (NHEJ) remains unclear. To better understand the degree to which alt-EJ depends on annealing at pre-existing microhomologies, we examined inaccurate repair of an I-SceI DSB lacking nearby microhomologies of greater than four nucleotides in Drosophila. Lig4 deficiency affected neither frequency nor length of junctional microhomology, but significantly increased insertion frequency. Many insertions appeared to be templated. Based on sequence analysis of repair junctions, we propose a model of synthesis-dependent microhomology-mediated end joining (SD-MMEJ), in which de novo synthesis by an accurate non-processive DNA polymerase creates microhomology. Repair junctions with apparent blunt joins, junctional microhomologies and short indels (deletion with insertion) are often considered to reflect different repair mechanisms. However, a majority of each type had structures consistent with the predictions of our SD-MMEJ model. This suggests that a single underlying mechanism could be responsible for all three repair product types. Genetic analysis indicates that SD-MMEJ is Ku70, Lig4 and Rad51-independent but impaired in mus308 (POLQ) mutants.

The BLM helicase is a member of the RecQ DNA helicase family and is mutated in the cancer-prone disorder Bloom syndrome. BLM plays a role in a number of cellular processes including DNA double-strand break repair, Holliday junction dissolution, and chromosome segregation. In Drosophila melanogaster, the BLM ortholog (DmBlm) is encoded by the mus309 gene. To study the role of DmBlm in double-strand break repair, we utilized a genetic assay in which a targeted DNA double-strand gap is created through excision of a P transposable element. By recovering and molecularly analyzing individual repair products from wild-type and mus309 male pre-meiotic germline cells, we demonstrated that the DmBlm helicase is involved in homologous recombination downstream of strand invasion. This assay can be adapted to test the roles of numerous DNA metabolic factors in DNA double-strand gap repair.

DNA interstrand crosslinks (ICLs) are complex lesions that covalently link both strands of the DNA double helix and impede essential cellular processes such as DNA replication and transcription. Recent studies suggest that multiple repair pathways are involved in their removal. Elegant genetic analysis has demonstrated that at least three distinct sets of pathways cooperate in the repair and/or bypass of ICLs in budding yeast. Although the mechanisms of ICL repair in mammals appear similar to those in yeast, important differences have been documented. In addition, mammalian crosslink repair requires other repair factors, such as the Fanconi anemia proteins, whose functions are poorly understood. Because many of these proteins are conserved in simpler metazoans, nonmammalian models have become attractive systems for studying the function(s) of key crosslink repair factors. This review discusses the contributions that various model organisms have made to the field of ICL repair. Specifically, it highlights how studies performed with C. elegans, Drosophila, Xenopus, and the social amoeba Dictyostelium serve to complement those from bacteria, yeast, and mammals. Together, these investigations have revealed that although the underlying themes of ICL repair are largely conserved, the complement of DNA repair proteins utilized and the ways in which each of the proteins is used can vary substantially between different organisms.

Transposable elements are frequently used in Drosophila melanogaster for imprecise excision screens to delete genes of interest. However, these screens are highly variable in the number and size of deletions that are recovered. Here, we show that conducting excision screens in mus309 mutant flies that lack DmBlm, the Drosophila ortholog of the Bloom syndrome protein, increases the percentage and overall size of flanking deletions recovered after excision of either P or Minos elements.

The BLM helicase is a member of the RecQ DNA helicase family and is mutated in the cancer-prone disorder Bloom syndrome. BLM plays a role in a number of cellular processes including DNA double-strand break repair, Holliday junction dissolution, and chromosome segregation. In Drosophila melanogaster, the BLM ortholog (DmBlm) is encoded by the mus309 gene. To study the role of DmBlm in double-strand break repair, we utilized a genetic assay in which a targeted DNA double-strand gap is created through excision of a P transposable element. By recovering and molecularly analyzing individual repair products from wild-type and mus309 male pre-meiotic germline cells, we demonstrated that the DmBlm helicase is involved in homologous recombination downstream of strand invasion. This assay can be adapted to test the roles of numerous DNA metabolic factors in DNA double-strand gap repair.

DNA double-strand breaks are normal consequences of cell division and differentiation and must be repaired faithfully to maintain genome stability. Two mechanistically distinct pathways are known to efficiently repair double-strand breaks: homologous recombination and Ku-dependent non-homologous end joining. Recently, a third, less characterized repair mechanism, named microhomology-mediated end joining (MMEJ), has received increasing attention. MMEJ repairs DNA breaks via the use of substantial microhomology and always results in deletions. Furthermore, it probably contributes to oncogenic chromosome rearrangements and genetic variation in humans. Here, we summarize the genetic attributes of MMEJ from several model systems and discuss the relationship between MMEJ and 'alternative end joining'. We propose a mechanistic model for MMEJ and highlight important questions for future research.

Bloom Syndrome, a rare human disorder characterized by genomic instability and predisposition to cancer, is caused by mutation of BLM, which encodes a RecQ-family DNA helicase. The Drosophila melanogaster ortholog of BLM, DmBlm, is encoded by mus309. Mutations in mus309 cause hypersensitivity to DNA-damaging agents, female sterility, and defects in repairing double-strand breaks (DSBs). To better understand these phenotypes, we isolated novel mus309 alleles. Mutations that delete the N terminus of DmBlm, but not the helicase domain, have DSB repair defects as severe as those caused by null mutations. We found that female sterility is due to a requirement for DmBlm in early embryonic cell cycles; embryos lacking maternally derived DmBlm have anaphase bridges and other mitotic defects. These defects were less severe for the N-terminal deletion alleles, so we used one of these mutations to assay meiotic recombination. Crossovers were decreased to about half the normal rate, and the remaining crossovers were evenly distributed along the chromosome. We also found that spontaneous mitotic crossovers are increased by several orders of magnitude in mus309 mutants. These results demonstrate that DmBlm functions in multiple cellular contexts to promote genome stability.

Bloom syndrome is a rare disorder associated with cancer predisposition and genomic instability and is caused by loss of the RecQ helicase BLM. The Drosophila ortholog of BLM (DmBlm) is required for accurate repair of DNA double-strand gaps by homologous recombination. Repair products from DmBlm mutants have shorter repair synthesis tract lengths compared to wild type and are frequently associated with deletions flanking the break site. To determine the mechanisms responsible for deletion formation in the absence of DmBlm, we characterized repair after excision of the P { w a } element in various genetic backgrounds. Flies lacking DmRad51 do not have an elevated deletion frequency. Moreover, loss of DmRad51 suppresses deletion formation in DmBlm mutants. These data support a model in which DmBlm acts downstream of strand invasion to unwind a D-loop intermediate to free the newly synthesized strand. In the absence of DmBlm, alternative pathways of D-loop disassembly result in short repair synthesis tracts or flanking deletions. This model explains how RecQ helicases can promote homologous recombination while preventing illegitimate recombination.