Cross-species investigation into the requirement of XPA for nucleotide excision repair.

After reconstitution of nucleotide excision repair (excision repair) with XPA, RPA, XPC, TFIIH, XPF-ERCC1 and XPG, it was concluded that these six factors are the minimal essential components of the excision repair machinery. All six factors are highly conserved across diverse organisms spanning yeast to humans, yet no identifiable homolog of the XPA gene exists in many eukaryotes including green plants. Nevertheless, excision repair is reported to be robust in the XPA-lacking organism, Arabidopsis thaliana, which raises a fundamental question of whether excision repair could occur without XPA in other organisms. Here, we performed a phylogenetic analysis of XPA across all species with annotated genomes and then quantitatively measured excision repair in the absence of XPA using the sensitive whole-genome qXR-Seq method in human cell lines and two model organisms, Caenorhabditis elegans and Drosophila melanogaster. We find that although the absence of XPA results in inefficient excision repair and UV-sensitivity in humans, flies, and worms, excision repair of UV-induced DNA damage is detectable over background. These studies have yielded a significant discovery regarding the evolution of XPA protein and its mechanistic role in nucleotide excision repair.

CSB-independent, XPC-dependent transcription-coupled repair in Drosophila.

Drosophila melanogaster has been extensively used as a model system to study ionizing radiation and chemical-induced mutagenesis, double-strand break repair, and recombination. However, there are only limited studies on nucleotide excision repair in this important model organism. An early study reported that Drosophila lacks the transcription-coupled repair (TCR) form of nucleotide excision repair. This conclusion was seemingly supported by the Drosophila genome sequencing project, which revealed that Drosophila lacks a homolog to CSB, which is known to be required for TCR in mammals and yeasts. However, by using excision repair sequencing (XR-seq) genome-wide repair mapping technology, we recently found that the Drosophila S2 cell line performs TCR comparable to human cells. Here, we have extended this work to Drosophila at all its developmental stages. We find TCR takes place throughout the life cycle of the organism. Moreover, we find that in contrast to humans and other multicellular organisms previously studied, the XPC repair factor is required for both global and transcription-coupled repair in Drosophila.

DNA polymerase theta suppresses mitotic crossing over.

Polymerase theta-mediated end joining (TMEJ) is a chromosome break repair pathway that is able to rescue the lethality associated with the loss of proteins involved in early steps in homologous recombination (e.g., BRCA1/2). This is due to the ability of polymerase theta (Pol θ) to use resected, 3' single stranded DNA tails to repair chromosome breaks. These resected DNA tails are also the starting substrate for homologous recombination. However, it remains unknown if TMEJ can compensate for the loss of proteins involved in more downstream steps during homologous recombination. Here we show that the Holliday junction resolvases SLX4 and GEN1 are required for viability in the absence of Pol θ in Drosophila melanogaster, and lack of all three proteins results in high levels of apoptosis. Flies deficient in Pol θ and SLX4 are extremely sensitive to DNA damaging agents, and mammalian cells require either Pol θ or SLX4 to survive. Our results suggest that TMEJ and Holliday junction formation/resolution share a common DNA substrate, likely a homologous recombination intermediate, that when left unrepaired leads to cell death. One major consequence of Holliday junction resolution by SLX4 and GEN1 is cancer-causing loss of heterozygosity due to mitotic crossing over. We measured mitotic crossovers in flies after a Cas9-induced chromosome break, and observed that this mutagenic form of repair is increased in the absence of Pol θ. This demonstrates that TMEJ can function upstream of the Holiday junction resolvases to protect cells from loss of heterozygosity. Our work argues that Pol θ can thus compensate for the loss of the Holliday junction resolvases by using homologous recombination intermediates, suppressing mitotic crossing over and preserving the genomic stability of cells.

A pathway for error-free non-homologous end joining of resected meiotic double-strand breaks.

Programmed DNA double-strand breaks (DSBs) made during meiosis are repaired by recombination with the homologous chromosome to generate, at selected sites, reciprocal crossovers that are critical for the proper separation of homologs in the first meiotic division. Backup repair processes can compensate when the normal meiotic recombination processes are non-functional. We describe a novel backup repair mechanism that occurs when the homologous chromosome is not available in Drosophila melanogaster meiosis. In the presence of a previously described mutation (Mcm5A7) that disrupts chromosome pairing, DSB repair is initiated by homologous recombination but is completed by non-homologous end joining (NHEJ). Remarkably, this process yields precise repair products. Our results provide support for a recombination intermediate recently proposed in mouse meiosis, in which an oligonucleotide bound to the Spo11 protein that catalyzes DSB formation remains bound after resection. We propose that this oligonucleotide functions as a primer for fill-in synthesis to allow scarless repair by NHEJ. We argue that this is a conserved repair mechanism that is likely to be invoked to overcome occasional challenges in normal meiosis.

Mechanistic basis for microhomology identification and genome scarring by polymerase theta.

DNA polymerase theta mediates an end joining pathway (TMEJ) that repairs chromosome breaks. It requires resection of broken ends to generate long, 3' single-stranded DNA tails, annealing of complementary sequence segments (microhomologies) in these tails, followed by microhomology-primed synthesis sufficient to resolve broken ends. The means by which microhomologies are identified is thus a critical step in this pathway, but is not understood. Here we show microhomologies are identified by a scanning mechanism initiated from the 3' terminus and favoring bidirectional progression into flanking DNA, typically to a maximum of 15 nucleotides into each flank. Polymerase theta is frequently insufficiently processive to complete repair of breaks in microhomology-poor, AT-rich regions. Aborted synthesis leads to one or more additional rounds of microhomology search, annealing, and synthesis; this promotes complete repair in part because earlier rounds of synthesis generate microhomologies de novo that are sufficiently long that synthesis is more processive. Aborted rounds of synthesis are evident in characteristic genomic scars as insertions of 3 to 30 bp of sequence that is identical to flanking DNA ("templated" insertions). Templated insertions are present at higher levels in breast cancer genomes from patients with germline BRCA1/2 mutations, consistent with an addiction to TMEJ in these cancers. Our work thus describes the mechanism for microhomology identification and shows how it both mitigates limitations implicit in the microhomology requirement and generates distinctive genomic scars associated with pathogenic genome instability.

Centromeric SMC1 promotes centromere clustering and stabilizes meiotic homolog pairing.

During meiosis, each chromosome must selectively pair and synapse with its own unique homolog to enable crossover formation and subsequent segregation. How homolog pairing is maintained in early meiosis to ensure synapsis occurs exclusively between homologs is unknown. We aimed to further understand this process by examining the meiotic defects of a unique Drosophila mutant, Mcm5A7. We found that Mcm5A7 mutants are proficient in homolog pairing at meiotic onset yet fail to maintain pairing as meiotic synapsis ensues, causing seemingly normal synapsis between non-homologous loci. This pairing defect corresponds with a reduction of SMC1-dependent centromere clustering at meiotic onset. Overexpressing SMC1 in this mutant significantly restores centromere clustering, homolog pairing, and crossover formation. These data indicate that the initial meiotic pairing of homologs is not sufficient to yield synapsis exclusively between homologs and provide a model in which meiotic homolog pairing must be stabilized by centromeric SMC1 to ensure proper synapsis.

Bloom syndrome helicase in meiosis: Pro-crossover functions of an anti-crossover protein.

The functions of the Bloom syndrome helicase (BLM) and its orthologs are well characterized in mitotic DNA damage repair, but their roles within the context of meiotic recombination are less clear. In meiotic recombination, multiple repair pathways are used to repair meiotic DSBs, and current studies suggest that BLM may regulate the use of these pathways. Based on literature from Saccharomyces cerevisiae, Arabidopsis thaliana, Mus musculus, Drosophila melanogaster, and Caenorhabditis elegans, we present a unified model for a critical meiotic role of BLM and its orthologs. In this model, BLM and its orthologs utilize helicase activity to regulate the use of various pathways in meiotic recombination by continuously disassembling recombination intermediates. This unwinding activity provides the meiotic program with a steady pool of early recombination substrates, increasing the probability for a DSB to be processed by the appropriate pathway. As a result of BLM activity, crossovers are properly placed throughout the genome, promoting proper chromosomal disjunction at the end of meiosis. This unified model can be used to further refine the complex role of BLM and its orthologs in meiotic recombination.

Substrate preference of Gen endonucleases highlights the importance of branched structures as DNA damage repair intermediates.

Human GEN1 and yeast Yen1 are endonucleases with the ability to cleave Holliday junctions (HJs), which are proposed intermediates in recombination. In vivo, GEN1 and Yen1 function secondarily to Mus81, which has weak activity on intact HJs. We show that the genetic relationship is reversed in Drosophila, with Gen mutants having more severe defects than mus81 mutants. In vitro, DmGen, like HsGEN1, efficiently cleaves HJs, 5΄ flaps, splayed arms, and replication fork structures. We find that the cleavage rates for 5΄ flaps are significantly higher than those for HJs for both DmGen and HsGEN1, even in vast excess of enzyme over substrate. Kinetic studies suggest that the difference in cleavage rates results from a slow, rate-limiting conformational change prior to HJ cleavage: formation of a productive dimer on the HJ. Despite the stark difference in vivo that Drosophila uses Gen over Mus81 and humans use MUS81 over GEN1, we find the in vitro activities of DmGen and HsGEN1 to be strikingly similar. These findings suggest that simpler branched structures may be more important substrates for Gen orthologs in vivo, and highlight the utility of using the Drosophila model system to further understand these enzymes.

Drosophila MUS312 and the vertebrate ortholog BTBD12 interact with DNA structure-specific endonucleases in DNA repair and recombination.

DNA recombination and repair pathways require structure-specific endonucleases to process DNA structures that include forks, flaps, and Holliday junctions. Previously, we determined that the Drosophila MEI-9-ERCC1 endonuclease interacts with the MUS312 protein to produce meiotic crossovers, and that MUS312 has a MEI-9-independent role in interstrand crosslink (ICL) repair. The importance of MUS312 to pathways crucial for maintaining genomic stability in Drosophila prompted us to search for orthologs in other organisms. Based on sequence, expression pattern, conserved protein-protein interactions, and ICL repair function, we determined that the mammalian ortholog of MUS312 is BTBD12. Orthology between these proteins and S. cerevisiae Slx4 helped identify a conserved interaction with a second structure-specific endonuclease, SLX1. Genetic and biochemical evidence described here and in related papers suggest that MUS312 and BTBD12 direct Holliday junction resolution by at least two distinct endonucleases in different recombination and repair contexts.

Eliminating both canonical and short-patch mismatch repair in Drosophila melanogaster suggests a new meiotic recombination model.

In most meiotic systems, recombination is essential to form connections between homologs that ensure their accurate segregation from one another. Meiotic recombination is initiated by DNA double-strand breaks that are repaired using the homologous chromosome as a template. Studies of recombination in budding yeast have led to a model in which most early repair intermediates are disassembled to produce noncrossovers. Selected repair events are stabilized so they can proceed to form double-Holliday junction (dHJ) intermediates, which are subsequently resolved into crossovers. This model is supported in yeast by physical isolation of recombination intermediates, but the extent to which it pertains to animals is unknown. We sought to test this model in Drosophila melanogaster by analyzing patterns of heteroduplex DNA (hDNA) in recombination products. Previous attempts to do this have relied on knocking out the canonical mismatch repair (MMR) pathway, but in both yeast and Drosophila the resulting recombination products are complex and difficult to interpret. We show that, in Drosophila, this complexity results from a secondary, short-patch MMR pathway that requires nucleotide excision repair. Knocking out both canonical and short-patch MMR reveals hDNA patterns that reveal that many noncrossovers arise after both ends of the break have engaged with the homolog. Patterns of hDNA in crossovers could be explained by biased resolution of a dHJ; however, considering the noncrossover and crossover results together suggests a model in which a two-end engagement intermediate with unligated HJs can be disassembled by a helicase to a produce noncrossover or nicked by a nuclease to produce a crossover. While some aspects of this model are similar to the model from budding yeast, production of both noncrossovers and crossovers from a single, late intermediate is a fundamental difference that has important implications for crossover control.

Three structure-selective endonucleases are essential in the absence of BLM helicase in Drosophila.

DNA repair mechanisms in mitotically proliferating cells avoid generating crossovers, which can contribute to genome instability. Most models for the production of crossovers involve an intermediate with one or more four-stranded Holliday junctions (HJs), which are resolved into duplex molecules through cleavage by specialized endonucleases. In vitro studies have implicated three nuclear enzymes in HJ resolution: MUS81-EME1/Mms4, GEN1/Yen1, and SLX4-SLX1. The Bloom syndrome helicase, BLM, plays key roles in preventing mitotic crossover, either by blocking the formation of HJ intermediates or by removing HJs without cleavage. Saccharomyces cerevisiae mutants that lack Sgs1 (the BLM ortholog) and either Mus81-Mms4 or Slx4-Slx1 are inviable, but mutants that lack Sgs1 and Yen1 are viable. The current view is that Yen1 serves primarily as a backup to Mus81-Mms4. Previous studies with Drosophila melanogaster showed that, as in yeast, loss of both DmBLM and MUS81 or MUS312 (the ortholog of SLX4) is lethal. We have now recovered and analyzed mutations in Drosophila Gen. As in yeast, there is some redundancy between Gen and mus81; however, in contrast to the case in yeast, GEN plays a more predominant role in responding to DNA damage than MUS81-MMS4. Furthermore, loss of DmBLM and GEN leads to lethality early in development. We present a comparison of phenotypes occurring in double mutants that lack DmBLM and either MUS81, GEN, or MUS312, including chromosome instability and deficiencies in cell proliferation. Our studies of synthetic lethality provide insights into the multiple functions of DmBLM and how various endonucleases may function when DmBLM is absent.

Centromere-proximal suppression of meiotic crossovers in Drosophila is robust to changes in centromere number, repetitive DNA content, and centromere-clustering.

Accurate segregation of homologous chromosomes during meiosis depends on both the presence and the regulated placement of crossovers (COs). The centromere effect, or CO exclusion in pericentromeric regions of the chromosome, is a meiotic CO patterning phenomenon that helps prevent nondisjunction, thereby protecting against chromosomal disorders and other meiotic defects. Despite being identified nearly a century ago, the mechanisms behind this fundamental cellular process remain unknown, with most studies of the Drosophila centromere effect focusing on local influences of the centromere and pericentric heterochromatin. In this study, we sought to investigate whether dosage changes in centromere number and repetitive DNA content affect the strength of the centromere effect, using phenotypic recombination mapping. Additionally, we studied the effects of repetitive DNA function on centromere effect strength using satellite DNA-binding protein mutants displaying defective centromere-clustering in meiotic nuclei. Despite what previous studies suggest, our results show that the Drosophila centromere effect is robust to changes in centromere number, repetitive DNA content, as well as repetitive DNA function. Our study suggests that the centromere effect is unlikely to be spatially controlled, providing novel insight into the mechanisms behind the Drosophila centromere effect.

Functions of the Bloom Syndrome Helicase N-terminal Intrinsically Disordered Region.

Bloom Syndrome helicase (Blm) is a RecQ family helicase involved in DNA repair, cell-cycle progression, and development. Pathogenic variants in human BLM cause the autosomal recessive disorder Bloom Syndrome, characterized by predisposition to numerous types of cancer. Prior studies of Drosophila Blm mutants lacking helicase activity or protein have shown sensitivity to DNA damaging agents, defects in repairing DNA double-strand breaks (DSBs), female sterility, and improper segregation of chromosomes in meiosis. Blm orthologs have a well conserved and highly structured RecQ helicase domain, but more than half of the protein, particularly in the N-terminus, is predicted to be unstructured. Because this region is poorly conserved across multicellular organisms, we compared closely related species to identify regions of conservation, potentially indicating important functions. We deleted two of these Drosophila-conserved regions in D. melanogaster using CRISPR/Cas9 gene editing and assessed the effects on different Blm functions. Each deletion had distinct effects on different Blm activities. Deletion of either conserved region 1 (CR1) or conserved region 2 (CR2) compromised DSB repair through synthesis-dependent strand annealing and resulted in increased mitotic crossovers. In contrast, CR2 is critical for embryonic development but CR1 is not as important. CR1 deletion allows for proficient meiotic chromosome segregation but does lead to defects in meiotic crossover designation and patterning. Finally, deletion of CR2 does not lead to significant meiotic defects, indicating that while each region has overlapping functions, there are discreet roles facilitated by each. These results provide novel insights into functions of the N-terminal disordered region of Blm.

Centromere-Proximal Suppression of Meiotic Crossovers in Drosophila is Robust to Changes in Centromere Number and Repetitive DNA Content.

Accurate segregation of homologous chromosomes during meiosis depends on both the presence and regulated placement of crossovers (COs). The centromere effect (CE), or CO exclusion in pericentromeric regions of the chromosome, is a meiotic CO patterning phenomenon that helps prevent nondisjunction (NDJ), thereby protecting against chromosomal disorders and other meiotic defects. Despite being identified nearly a century ago, the mechanisms behind this fundamental cellular process remain unknown, with most studies of the Drosophila CE focusing on local influences of the centromere and pericentric heterochromatin. In this study, we sought to investigate whether dosage changes in centromere number and repetitive DNA content affect the strength of the CE, using phenotypic recombination mapping. Additionally, we also studied the effects of repetitive DNA function on CE strength using satellite-DNA binding protein mutants shown to have defective centromere clustering. Despite what previous studies suggest, our results show that the Drosophila CE is robust to dosage changes in centromere number and repetitive DNA content, and potentially also to repetitive DNA function. Our study suggests that the CE is unlikely to be spatially controlled, providing novel insight into the mechanisms behind the Drosophila centromere effect.

ATM/Chk2 and ATR/Chk1 Pathways Respond to DNA Damage Induced by Movento® 240SC and Envidor® 240SC Keto-Enol Insecticides in the Germarium of Drosophila melanogaster.

DNA damage response (DDR) pathways in keto-enol genotoxicity have not been characterized, and few studies have reported genotoxic effects in non-target organisms. The present study shows that concentrations of 11.2, 22.4, 37.3 mg/L of Movento® 240SC and 12.3, 24.6, 41.1 mg/L of Envidor® 240SC for 72 h oral exposure induced DSBs by significantly increasing the percentage of γH2AV expression in regions 2b and 3 from the germarium of wild type females of Drosophila melanogaster Oregon R, compared to the control group (0.0 mg/L of insecticides), via confocal immunofluorescence microscopy. The comparison between both insecticides' reveals that only the Envidor® 240SC induces concentration-dependent DNA damage, as well as structural changes in the germarium. We determined that the DDR induced by Movento® 240SC depends on the activation of the ATMtefu, Chk1grp and Chk2lok kinases by significantly increasing the percentage of expression of γH2AV in regions 2b and 3 of the germarium, and that ATRmei-29D and p53dp53 kinases only respond at the highest concentration of 37.3 mg/L of Movento® 240SC. With the Envidor® 240SC insecticide, we determined that the DDR depends on the activation of the ATRmei-29D/Chk1grp and ATMtefu/Chk2lok kinases, and p53dp53 by significantly increasing the percentage of expression of γH2AV in the germarium.

The effect of repeat length on Marcal1-dependent single-strand annealing in Drosophila.

Proper repair of DNA double-strand breaks is essential to the maintenance of genomic stability and avoidance of genetic disease. Organisms have many ways of repairing double-strand breaks, including the use of homologous sequences through homology-directed repair. While homology-directed repair is often error free, in single-strand annealing homologous repeats flanking a double-strand break are annealed to one another, leading to the deletion of one repeat and the intervening sequences. Studies in yeast have shown a relationship between the length of the repeat and single-strand annealing efficacy. We sought to determine the effects of homology length on single-strand annealing in Drosophila, as Drosophila uses a different annealing enzyme (Marcal1) than yeast. Using an in vivo single-strand annealing assay, we show that 50 base pairs are insufficient to promote single-strand annealing and that 500-2,000 base pairs are required for maximum efficiency. Loss of Marcal1 generally followed the same homology length trend as wild-type flies, with single-strand annealing frequencies reduced to about a third of wild-type frequencies regardless of homology length. Interestingly, we find a difference in single-strand annealing rates between 500-base pair homologies that align to the annealing target either nearer or further from the double-strand break, a phenomenon that may be explained by Marcal1 dynamics. This study gives insights into Marcal1 function and provides important information to guide the design of genome engineering strategies that use single-strand annealing to integrate linear DNA constructs into a chromosomal double-strand break.

Meiotic versus mitotic recombination: two different routes for double-strand break repair: the different functions of meiotic versus mitotic DSB repair are reflected in different pathway usage and different outcomes.

Studies in the yeast Saccharomyces cerevisiae have validated the major features of the double-strand break repair (DSBR) model as an accurate representation of the pathway through which meiotic crossovers (COs) are produced. This success has led to this model being invoked to explain double-strand break (DSB) repair in other contexts. However, most non-crossover (NCO) recombinants generated during S. cerevisiae meiosis do not arise via a DSBR pathway. Furthermore, it is becoming increasingly clear that DSBR is a minor pathway for recombinational repair of DSBs that occur in mitotically-proliferating cells and that the synthesis-dependent strand annealing (SDSA) model appears to describe mitotic DSB repair more accurately. Fundamental dissimilarities between meiotic and mitotic recombination are not unexpected, since meiotic recombination serves a very different purpose (accurate chromosome segregation, which requires COs) than mitotic recombination (repair of DNA damage, which typically generates NCOs).

Blm helicase facilitates rapid replication of repetitive DNA sequences in early Drosophila development.

The absence of functional BLM DNA helicase, a member of the RecQ family of helicases, is responsible for the rare human disorder Bloom Syndrome, which results in developmental abnormalities, DNA repair defects, genomic instability, and a predisposition to cancer. In Drosophila melanogaster, the orthologous Blm protein is essential during early development when the embryo is under the control of maternal gene products. We show that lack of functional maternal Blm during the syncytial cell cycles of Drosophila embryonic development results in severe nuclear defects and lethality. Amongst the small fraction of embryos from Blm mutant mothers that survive to adulthood, a prominent sex-bias favors the class that inherits less repetitive DNA content, which serves as an endogenous source of replication stress. This selection against repetitive DNA content reflects a role for Blm in facilitating replication through repetitive sequences during the rapid S-phases of syncytial cell cycles. During these syncytial cycles, Blm is not required for complex DNA double-strand break repair; however, the progeny sex-bias resulting from the absence of maternal Blm is exacerbated by repetitive DNA sequences and by the slowing of replication fork progression, suggesting that the essential role for Blm during this stage is to manage replication fork stress brought about by impediments to fork progression. Additionally, our data suggest that Blm is only required to manage this replication stress during embryonic development, and likely only during the early, rapid syncytial cell cycles, and not at later developmental stages. These results provide novel insights into Blm function throughout development.

Interstrand crosslink repair: can XPF-ERCC1 be let off the hook?

The interstrand crosslink (ICL) presents a challenge to both the cell and the scientist. From a clinical standpoint, these lesions are particularly intriguing: ICL-inducing agents are powerful tools in cancer chemotherapy, and spontaneous ICLs have recently been linked with accelerated aging phenotypes. Nevertheless, the ICL repair process has proven difficult to elucidate. Here we discuss recent additions to the current model and argue that the endonuclease xeroderma pigmentosum complementation group F-excision repair cross-complementing rodent repair deficiency complementation group 1 (XPF-ERCC1) has been heretofore misplaced. During nucleotide excision repair, XPF-ERCC1 makes a single-strand nick adjacent to the lesion. XPF-ERCC1 has been thought to play an analogous role in ICL repair. However, recent data has implicated XPF-ERCC1 in homologous recombination. We suggest that this role, rather than its function in nucleotide excision repair, defines its importance to ICL repair.