Gene Conversion Tracts Research Articles

Genomics of natural populations: gene conversion events reveal selected genes within the inversions of Drosophila pseudoobscura.

When adaptive phenotypic variation or quantitative trait loci map within an inverted segment of a chromosome, researchers often despair because the suppression of crossing over will prevent the discovery of selective target genes that established the rearrangement. If an inversion polymorphism is old enough, then the accumulation of gene conversion tracts offers the promise that quantitative trait loci or selected loci within inversions can be mapped. The inversion polymorphism of Drosophila pseudoobscura is a model system to show that gene conversion analysis is a useful tool for mapping selected loci within inversions. D. pseudoobscura has over 30 different chromosomal arrangements on the third chromosome (Muller C) in natural populations and their frequencies vary with changes in environmental habitats. Statistical tests of five D. pseudoobscura gene arrangements identified outlier genes within inverted regions that had potentially heritable variation, either fixed amino acid differences or differential expression patterns. We use genome sequences of the inverted third chromosome (Muller C) to infer 98,443 gene conversion tracts for a total coverage of 142 Mb or 7.2× coverage of the 19.7 Mb chromosome. We estimated gene conversion tract coverage in the 2,668 genes on Muller C and tested whether gene conversion coverage was similar among arrangements for outlier vs non-outlier loci. Outlier genes had lower gene conversion tract coverage among arrangements than the non-outlier genes suggesting that selection removes exchanged DNA in the outlier genes. These data support the hypothesis that the third chromosome in D. pseudoobscura captured locally adapted combinations of alleles prior to inversion mutation events.

NCOurd: modelling length distributions of NCO events and gene conversion tracts.

Meiotic recombination is the main driving force of human genetic diversity, along with mutations. Recombinations split into crossovers, separating large chromosomal regions originating from different homologous chromosomes, and non-crossovers (NCOs), where a small segment from one chromosome is embedded in a region originating from the homologous chromosome. NCOs are much less studied than mutations and crossovers as NCOs are short and can only be detected at markers heterozygous in the transmitting parent, leaving most of them undetectable. The detectable NCOs, known as gene conversions, hide information about NCOs, including their number and length, waiting to be unveiled. We introduce NCOurd, software, and algorithm, based on an expectation-maximization algorithm, to estimate the number of NCOs and their length distribution from gene conversion data. https://github.com/DecodeGenetics/NCOurd.

Cdc14 phosphatase counteracts Cdk-dependent Dna2 phosphorylation to inhibit resection during recombinational DNA repair

Cyclin-dependent kinase (Cdk) stimulates resection of DNA double-strand breaks ends to generate single-stranded DNA (ssDNA) needed for recombinational DNA repair. Here we show in Saccharomyces cerevisiae that lack of the Cdk-counteracting phosphatase Cdc14 produces abnormally extended resected tracts at the DNA break ends, involving the phosphatase in the inhibition of resection. Over-resection in the absence of Cdc14 activity is bypassed when the exonuclease Dna2 is inactivated or when its Cdk consensus sites are mutated, indicating that the phosphatase restrains resection by acting through this nuclease. Accordingly, mitotically activated Cdc14 promotes Dna2 dephosphorylation to exclude it from the DNA lesion. Cdc14-dependent resection inhibition is essential to sustain DNA re-synthesis, thus ensuring the appropriate length, frequency, and distribution of the gene conversion tracts. These results establish a role for Cdc14 in controlling the extent of resection through Dna2 regulation and demonstrate that the accumulation of excessively long ssDNA affects the accurate repair of the broken DNA by homologous recombination.

Lessons from the meiotic recombination landscape of the ZMM deficient budding yeast Lachancea waltii.

Meiotic recombination is a driving force for genome evolution, deeply characterized in a few model species, notably in the budding yeast Saccharomyces cerevisiae. Interestingly, Zip2, Zip3, Zip4, Spo16, Msh4, and Msh5, members of the so-called ZMM pathway that implements the interfering meiotic crossover pathway in S. cerevisiae, have been lost in Lachancea yeast species after the divergence of Lachancea kluyveri from the rest of the clade. In this context, after investigating meiosis in L. kluyveri, we determined the meiotic recombination landscape of Lachancea waltii. Attempts to generate diploid strains with fully hybrid genomes invariably resulted in strains with frequent whole-chromosome aneuploidy and multiple extended regions of loss of heterozygosity (LOH), which mechanistic origin is so far unclear. Despite the lack of multiple ZMM pro-crossover factors in L. waltii, numbers of crossovers and noncrossovers per meiosis were higher than in L. kluyveri but lower than in S. cerevisiae, for comparable genome sizes. Similar to L. kluyveri but opposite to S. cerevisiae, L. waltii exhibits an elevated frequency of zero-crossover bivalents. Lengths of gene conversion tracts for both crossovers and non-crossovers in L. waltii were comparable to those observed in S. cerevisiae and shorter than in L. kluyveri despite the lack of Mlh2, a factor limiting conversion tract size in S. cerevisiae. L. waltii recombination hotspots were not shared with either S. cerevisiae or L. kluyveri, showing that meiotic recombination hotspots can evolve at a rather limited evolutionary scale within budding yeasts. Finally, L. waltii crossover interference was reduced relative to S. cerevisiae, with interference being detected only in the 25 kb distance range. Detection of positive inference only at short distance scales in the absence of multiple ZMM factors required for interference-sensitive crossovers in other systems likely reflects interference between early recombination precursors such as DSBs.

Haplotype structures and polymorphisms of dog leukocyte antigen (DLA) class I loci shaped by intralocus and interlocus recombination events.

The dog leukocyte antigen (DLA) class I genomic region is located on chromosome 12, and the class I genomic region is composed of at least two distinct haplotypic gene structures, DLA-88-DLA-12 and DLA-88-DLA-88L. However, detailed information of the genomic differences among DLA-88, DLA-12, and DLA-88L are still lacking at the full-length gene level, and therefore, DLA allelic sequences classified for each of these loci are limited in number so far. In this study, we determined the DNA sequence of a 95-kb DLA class I genomic region including DLA-88, DLA-12/88L, and DLA-64 with three DLA homozygous dogs and of 37 full-length allelic gene sequences for DLA-88 and DLA-12/88L loci in 26 DLA class I homozygous dogs. Nucleotide diversity profiles of the 95-kb regions and sequence identity scores of the allelic sequences suggested that DLA-88L is a hybrid gene generated by interlocus and/or intralocus gene conversion between DLA-88 and DLA-12. The putative minimum conversion tract was estimated to be at least an 850-bp segment in length located from the 5´flanking untranslated region to the end of intron 2. In addition, at least one DLA-12 allele (DLA-12*004:01) was newly generated by interlocus gene conversion. In conclusion, the analysis for the occurrence of gene conversion within the dog DLA class I region revealed intralocus gene conversion tracts in 17 of 27 DLA-88 alleles and two of 10 DLA-12 alleles, suggesting that intralocus gene conversion has played an important role in expanding DLA allelic variations.

HELQ is a dual-function DSB repair enzyme modulated by RPA and RAD51

DNA double-stranded breaks (DSBs) are deleterious lesions, and their incorrect repair can drive cancer development1. HELQ is a superfamily 2 helicase with 3′ to 5′ polarity, and its disruption in mice confers germ cells loss, infertility and increased predisposition to ovarian and pituitary tumours2–4. At the cellular level, defects in HELQ result in hypersensitivity to cisplatin and mitomycin C, and persistence of RAD51 foci after DNA damage3,5. Notably, HELQ binds to RPA and the RAD51-paralogue BCDX2 complex, but the relevance of these interactions and how HELQ functions in DSB repair remains unclear3,5,6. Here we show that HELQ helicase activity and a previously unappreciated DNA strand annealing function are differentially regulated by RPA and RAD51. Using biochemistry analyses and single-molecule imaging, we establish that RAD51 forms a complex with and strongly stimulates HELQ as it translocates during DNA unwinding. By contrast, RPA inhibits DNA unwinding by HELQ but strongly stimulates DNA strand annealing. Mechanistically, we show that HELQ possesses an intrinsic ability to capture RPA-bound DNA strands and then displace RPA to facilitate annealing of complementary sequences. Finally, we show that HELQ deficiency in cells compromises single-strand annealing and microhomology-mediated end-joining pathways and leads to bias towards long-tract gene conversion tracts during homologous recombination. Thus, our results implicate HELQ in multiple arms of DSB repair through co-factor-dependent modulation of intrinsic translocase and DNA strand annealing activities.

A role for synaptonemal complex in meiotic mismatch repair.

A large subset of meiotic recombination intermediates form within the physical context of synaptonemal complex (SC), but the functional relationship between SC structure and homologous recombination remains obscure. Our prior analysis of strains deficient for SC central element proteins demonstrated that tripartite SC is dispensable for interhomolog recombination in Saccharomyces cerevisiae. Here, we report that while dispensable for recombination per se, SC proteins promote efficient mismatch repair at interhomolog recombination sites. Failure to repair mismatches within heteroduplex-containing meiotic recombination intermediates leads to genotypically sectored colonies (postmeiotic segregation events). We discovered increased postmeiotic segregation at THR1 in cells lacking Ecm11 or Gmc2, or in the SC-deficient but recombination-proficient zip1[Δ21-163] mutant. High-throughput sequencing of octad meiotic products furthermore revealed a genome-wide increase in recombination events with unrepaired mismatches in ecm11 mutants relative to wildtype. Meiotic cells missing Ecm11 display longer gene conversion tracts, but tract length alone does not account for the higher frequency of unrepaired mismatches. Interestingly, the per-nucleotide mismatch frequency is elevated in ecm11 when analyzing all gene conversion tracts, but is similar between wildtype and ecm11 if considering only those events with unrepaired mismatches. Thus, in both wildtype and ecm11 strains a subset of recombination events is susceptible to a similar degree of inefficient mismatch repair, but in ecm11 mutants a larger fraction of events fall into this inefficient repair category. Finally, we observe elevated postmeiotic segregation at THR1 in mutants with a dual deficiency in MutSγ crossover recombination and SC assembly, but not in the mlh3 mutant, which lacks MutSγ crossovers but has abundant SC. We propose that SC structure promotes efficient mismatch repair of joint molecule recombination intermediates, and that absence of SC is the molecular basis for elevated postmeiotic segregation in both MutSγ crossover-proficient (ecm11, gmc2) and MutSγ crossover-deficient (msh4, zip3) strains.

The Role of Drosophila CtIP in Homology-Directed Repair of DNA Double-Strand Breaks.

DNA double-strand breaks (DSBs) are a particularly genotoxic type of DNA damage that can result in chromosomal aberrations. Thus, proper repair of DSBs is essential to maintaining genome integrity. DSBs can be repaired by non-homologous end joining (NHEJ), where ends are processed before joining through ligation. Alternatively, DSBs can be repaired through homology-directed repair, either by homologous recombination (HR) or single-strand annealing (SSA). Both types of homology-directed repair are initiated by DNA end resection. In cultured human cells, the protein CtIP has been shown to play a role in DNA end resection through its interactions with CDK, BRCA1, DNA2, and the MRN complex. To elucidate the role of CtIP in a multicellular context, CRISPR/Cas9 genome editing was used to create a DmCtIPΔ allele in Drosophila melanogaster. Using the DSB repair reporter assay direct repeat of white (DR-white), a two-fold decrease in HR in DmCtIPΔ/Δ mutants was observed when compared to heterozygous controls. However, analysis of HR gene conversion tracts (GCTs) suggests DmCtIP plays a minimal role in determining GCT length. To assess the function of DmCtIP on both short (~550 bp) and long (~3.6 kb) end resection, modified homology-directed SSA repair assays were implemented, resulting in a two-fold decrease in SSA repair in both short and extensive end resection requirements in the DmCtIPΔ/Δ mutants compared to heterozygote controls. Through these analyses, we affirmed the importance of end resection on DSB repair pathway choice in multicellular systems, described the function of DmCtIP in short and extensive DNA end resection, and determined the impact of end resection on GCT length during HR.

Analyzing Plant Gene Targeting Outcomes and Conversion Tracts with Nanopore Sequencing.

The high-throughput molecular analysis of gene targeting (GT) events is made technically challenging by the residual presetabce of donor molecules. Large donor molecules restrict primer placement, resulting in long amplicons that cannot be readily analyzed using standard NGS pipelines or qPCR-based approaches such as ddPCR. In plants, removal of excess donor is time and resource intensive, often requiring plant regeneration and weeks to months of effort. Here, we utilized Oxford Nanopore Amplicon Sequencing (ONAS) to bypass the limitations imposed by donor molecules with 1 kb of homology to the target and dissected GT outcomes at three loci in Nicotiana benthamia leaves. We developed a novel bioinformatic pipeline, Phased ANalysis of Genome Editing Amplicons (PANGEA), to reduce the effect of ONAS error on amplicon analysis and captured tens of thousands of somatic plant GT events. Additionally, PANGEA allowed us to collect thousands of GT conversion tracts 5 days after reagent delivery with no selection, revealing that most events utilized tracts less than 100 bp in length when incorporating an 18 bp or 3 bp insertion. These data demonstrate the usefulness of ONAS and PANGEA for plant GT analysis and provide a mechanistic basis for future plant GT optimization.

Characterization of Non-selected Intermolecular Gene Conversion in the Polyploid Haloarchaeon Haloferax volcanii.

Gene conversion is defined as the non-reciprocal transfer of genetic information from one site to a homologous, but not identical site of the genome. In prokaryotes, gene conversion can increase the variance of sequences, like in antigenic variation, but can also lead to a homogenization of sequences, like in the concerted evolution of multigene families. In contrast to these intramolecular mechanisms, the intermolecular gene conversion in polyploid prokaryotes, which leads to the equalization of the multiple genome copies, has hardly been studied. We have previously shown the intermolecular gene conversion in halophilic and methanogenic archaea is so efficient that it can be studied without selecting for conversion events. Here, we have established an approach to characterize unselected intermolecular gene conversion in Haloferax volcanii making use of two genes that encode enzymes involved in carotenoid biosynthesis. Heterozygous strains were generated by protoplast fusion, and gene conversion was quantified by phenotype analysis or/and PCR. It was verified that unselected gene conversion is extremely efficient and it was shown that gene conversion tracts are much longer than in antigenic variation or concerted evolution in bacteria. Two sites were nearly always co-converted when they were 600 bp apart, and more than 30% co-conversion even occurred when two sites were 5 kbp apart. The gene conversion frequency was independent from the extent of genome differences, and even a one nucleotide difference triggered conversion.

The Pif1 helicase is actively inhibited during meiotic recombination which restrains gene conversion tract length.

Meiotic recombination ensures proper chromosome segregation to form viable gametes and results in gene conversions events between homologs. Conversion tracts are shorter in meiosis than in mitotically dividing cells. This results at least in part from the binding of a complex, containing the Mer3 helicase and the MutLβ heterodimer, to meiotic recombination intermediates. The molecular actors inhibited by this complex are elusive. The Pif1 DNA helicase is known to stimulate DNA polymerase delta (Pol δ) -mediated DNA synthesis from D-loops, allowing long synthesis required for break-induced replication. We show that Pif1 is also recruited genome wide to meiotic DNA double-strand break (DSB) sites. We further show that Pif1, through its interaction with PCNA, is required for the long gene conversions observed in the absence of MutLβ recruitment to recombination sites. In vivo, Mer3 interacts with the PCNA clamp loader RFC, and in vitro, Mer3-MutLβ ensemble inhibits Pif1-stimulated D-loop extension by Pol δ and RFC-PCNA. Mechanistically, our results suggest that Mer3-MutLβ may compete with Pif1 for binding to RFC-PCNA. Taken together, our data show that Pif1’s activity that promotes meiotic DNA repair synthesis is restrained by the Mer3-MutLβ ensemble which in turn prevents long gene conversion tracts and possibly associated mutagenesis.

Mapping chromosomal instability induced by small-molecular therapeutics in a yeast model.

The yeast Saccharomyces cerevisiae has been widely used as a model system for studying the physiological and pharmacological action of small-molecular drugs. Here, a heterozygous diploid S. cerevisiae strain QSS4 was generated to determine whether drugs could induce chromosomal instability by determining the frequency of mitotic recombination. Using the combination of a custom SNP microarray and yeast screening system, the patterns of chromosomal instability induced by drugs were explored at the whole genome level in QSS4. We found that Zeocin (a member of the bleomycin family) treatment increased the rate of genomic alterations, including aneuploidy, loss of heterozygosity (LOH), and chromosomal rearrangement over a hundred-fold. Most recombination events are likely to be initiated by DNA double-stand breaks directly generated by Zeocin. Another remarkable finding is that G4-motifs and low GC regions were significantly underrepresented within the gene conversion tracts of Zeocin-induced LOH events, indicating that certain DNA regions are less preferred Zeocin-binding sites in vivo. This study provides a novel paradigm for evaluating genetic toxicity of small-molecular drugs using yeast models.

Mismatch tolerance during homologous recombination in mammalian cells

We investigated the homology dependency of recombination in thymidine kinase (tk)-deficient mouse fibroblasts. Cells were transfected with DNA constructs harboring a herpes tk gene (the “recipient”) rendered non-functional by an oligonucleotide containing the recognition site for endonuclease I-SceI. Constructs also contained a “donor” tk sequence that could restore function to the recipient gene through spontaneous gene conversion or via repair of a double-strand break (DSB) at the I-SceI site. Recombination events were recoverable by selection for tk-positive clones. Three different donors were used containing 16, 25, or 33 mismatches relative to the recipient. The mismatches were clustered, forming an interval of “homeology” relative to the recipient sequences. We show that when homeologous sequences were surrounded by high homology, mismatches were frequently included in gene conversion events. Notably, conversion tracts from spontaneous recombination included either all or none of the mismatches, suggesting that recombination must begin and end in high homology. This requirement was relaxed for events that occurred near an induced DSB, as a significant number of these latter conversion tracts had one end positioned within homeology. Knock-down of mismatch repair showed that incorporation of mismatches into gene conversion tracts can involve repair of mismatched heteroduplex intermediates, indicating that mismatch repair does not necessarily impede homeologous genetic exchange. Our results illustrate (1) genetic exchange between homeologous sequences in a mammalian genome is enabled by nearby homology, (2) proximity to a DSB impacts the homology requirements for where genetic exchange may begin and end, and (3) mismatch correction and previously documented anti-recombination activity are separable functions of the mismatch repair machinery in mammalian cells.

Role of Homology and DNA Double‐Strand Breaks in Initiation of Intrachromosomal Recombination in Mammalian Cells

As part of our continuing investigations into the mechanisms of intrachromosomal recombination in mammalian cells, we stably transfected thymidine kinase deficient mouse fibroblasts with a DNA construct, pKM1, harboring a herpes thymidine kinase (tk) gene rendered nonfunctional by insertion of an oligonucleotide containing the recognition site for endonuclease I‐SceI. We refer to this latter tk sequence as the “recipient.” The construct also contained a closely linked truncated “donor” tk sequence. The recipient and donor were positioned as direct repeats with respect to transcriptional orientation. The donor could potentially restore function to the recipient gene via recombination with the recipient, and such events could be provoked by induction of a double‐strand break (DSB) at the I‐SceI site in the recipient. Such DSB repair events were recoverable by selection for tk‐positive clones. The donor contained 33 mismatches relative to the recipient. The mismatches were clustered, forming a localized segment of DNA sequence displaying about 20% divergence relative to the recipient, and the mismatched segment was surrounded by regions of high homology. When the donor was aligned with the recipient in pKM1, the DSB site in the recipient aligned opposite the mismatched segment, allowing us to potentially capture recombinational repair events initiating between diverged sequences. Our earlier work demonstrated that mammalian cells effectively avoid recombination between 20% diverged sequences. In work presented here, we asked whether flanking regions of high homology would enable genetic exchange between highly diverged sequences or, instead, would rejection of exchange between diverged sequences remain unchanged. We found that by surrounding mismatches with high homology, suppression of recombination between diverged sequences was overcome. Strikingly, we recovered a high frequency of gene conversion tracts positioned entirely within the mismatched sequences. We infer that such events were enabled by homologous pairing interactions between sequences surrounding the site of strand invasion. Our results suggest that the search for high homology prior to recombination is not mediated by an invading DNA terminus and may actually be independent of DSB formation. Further, the homology that enabled the recovered recombination events was located at a discrete distance from the actual invading DNA terminus. In an extension of our studies, we designed two additional constructs, pCW1 and pCW2. These constructs were similar to pKM1 except the recipient sequence in each was disrupted by an 8bp XhoI linker insertion and each construct contained a I‐SceI recognition site positioned within vector sequences at a location outside of either the recipient or donor sequence. Cells transfected with constructs pCW1 and pCW2 are being used to further explore whether a chromosomal DSB can induce and/or influence the nature of genetic exchanges occurring at a discrete distance from the DSB when the genetic exchanges themselves do not directly repair the DSB. Progress addressing this latter issue is presented.Support or Funding InformationThis work was supported by grant MCB 1157416 from the National Science Foundation to ASW and BCW.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Impact of Homologous Recombination on Silent Chromatin in Saccharomyces cerevisiae.

Specialized chromatin domains repress transcription of genes within them and present a barrier to many DNA-protein interactions. Silent chromatin in the budding yeast Saccharomyces cerevisiae, akin to heterochromatin of metazoans and plants, inhibits transcription of PolII- and PolIII-transcribed genes, yet somehow grants access to proteins necessary for DNA transactions like replication and homologous recombination. In this study, we adapted a novel assay to detect even transient changes in the dynamics of transcriptional silencing at HML after it served as a template for homologous recombination. Homologous recombination specifically targeted to HML via double-strand-break formation at a homologous locus often led to transient loss of transcriptional silencing at HML Interestingly, many cells could template homology-directed repair at HML without an obligate loss of silencing, even in recombination events with extensive gene conversion tracts. In a population of cells that experienced silencing loss following recombination, transcription persisted for 2-3 hr after all double-strand breaks were repaired. mRNA levels from cells that experienced recombination-induced silencing loss did not approach the amount of mRNA seen in cells lacking transcriptional silencing. Thus, silencing loss at HML after homologous recombination was short-lived and limited.

High homology is not required at the site of strand invasion during recombinational double-strand break repair in mammalian chromosomes

We investigated the impact of sequence divergence on DNA double-strand break (DSB) repair occurring via recombination in cultured thymidine kinase deficient mouse fibroblasts. We stably transfected cells with a DNA construct harboring a herpes thymidine kinase (tk) gene (the “recipient”) rendered nonfunctional by insertion of an oligonucleotide containing the recognition site for endonuclease I-SceI. The construct also contained a closely linked truncated “donor” tk sequence. The donor could potentially restore function to the recipient gene via recombination provoked by induction of a DSB at the I-SceI site in the recipient. Repair events were recoverable by selection for tk-positive clones. The donor contained 33 mismatches relative to the recipient. The mismatches were clustered, forming a localized segment of DNA sequence displaying about 20% divergence relative to the recipient, and the mismatched segment was surrounded by regions of high homology. When the donor was aligned with the recipient, the DSB site in the recipient aligned opposite the mismatched segment, allowing us to potentially capture recombinational repair events initiating between diverged sequences. Previous work demonstrated that mammalian cells effectively avoid recombination between 20% diverged sequences. In the current study we asked whether flanking regions of high homology would enable genetic exchange between highly diverged sequences or, instead, would rejection of exchange between diverged sequences remain unchanged. We found that by surrounding mismatches with high homology, suppression of recombination between diverged sequences was overcome. Strikingly, we recovered a high frequency of gene conversion tracts positioned entirely within the mismatched sequences. We infer that such events were enabled by homologous pairing interactions between sequences surrounding the site of strand invasion. Our results suggest a search for high homology prior to recombination that is not mediated by an invading DNA terminus.

The Role of Blm Helicase in Homologous Recombination, Gene Conversion Tract Length, and Recombination Between Diverged Sequences in Drosophilamelanogaster.

DNA double-strand breaks (DSBs) are a particularly deleterious class of DNA damage that threatens genome integrity. DSBs are repaired by three pathways: nonhomologous-end joining (NHEJ), homologous recombination (HR), and single-strand annealing (SSA). Drosophila melanogaster Blm (DmBlm) is the ortholog of Saccharomyces cerevisiae SGS1 and human BLM, and has been shown to suppress crossovers in mitotic cells and repair mitotic DNA gaps via HR. To further elucidate the role of DmBlm in repair of a simple DSB, and in particular recombination mechanisms, we utilized the Direct Repeat of white (DR-white) and Direct Repeat of whitewith mutations (DR-white.mu) repair assays in multiple mutant allele backgrounds. DmBlm null and helicase-dead mutants both demonstrated a decrease in repair by noncrossover HR, and a concurrent increase in non-HR events, possibly including SSA, crossovers, deletions, and NHEJ, although detectable processing of the ends was not significantly impacted. Interestingly, gene conversion tract lengths of HR repair events were substantially shorter in DmBlm null but not helicase-dead mutants, compared to heterozygote controls. Using DR-white.mu, we found that, in contrast to Sgs1, DmBlm is not required for suppression of recombination between diverged sequences. Taken together, our data suggest that DmBlm helicase function plays a role in HR, and the steps that contribute to determining gene conversion tract length are helicase-independent.

Mitotic Gene Conversion Tracts Associated with Repair of a Defined Double-Strand Break in Saccharomyces cerevisiae.

Mitotic recombination between homologous chromosomes leads to the uncovering of recessive alleles through loss of heterozygosity. In the current study, a defined double-strand break was used to initiate reciprocal loss of heterozygosity between diverged homologs of chromosome IV in Saccharomyces cerevisiae These events resulted from the repair of two broken chromatids, one of which was repaired as a crossover and the other as a noncrossover. Associated gene conversion tracts resulting from the donor-directed repair of mismatches formed during strand exchange (heteroduplex DNA) were mapped using microarrays. Gene conversion tracts associated with individual crossover and noncrossover events were similar in size and position, with half of the tracts being unidirectional and mapping to only one side of the initiating break. Among crossover events, this likely reflected gene conversion on only one side of the break, with restoration-type repair occurring on the other side. For noncrossover events, an ectopic system was used to directly compare gene conversion tracts produced in a wild-type strain to heteroduplex DNA tracts generated in the absence of the Mlh1 mismatch-repair protein. There was a strong bias for unidirectional tracts in the absence, but not in the presence, of Mlh1 This suggests that mismatch repair acts on heteroduplex DNA that is only transiently present in noncrossover intermediates of the synthesis dependent strand annealing pathway. Although the molecular features of events associated with loss of heterozygosity generally agreed with those predicted by current recombination models, there were unexpected complexities in associated gene conversion tracts.

Concerted action of the MutLβ heterodimer and Mer3 helicase regulates the global extent of meiotic gene conversion.

Gene conversions resulting from meiotic recombination are critical in shaping genome diversification and evolution. How the extent of gene conversions is regulated is unknown. Here we show that the budding yeast mismatch repair related MutLβ complex, Mlh1-Mlh2, specifically interacts with the conserved meiotic Mer3 helicase, which recruits it to recombination hotspots, independently of mismatch recognition. This recruitment is essential to limit gene conversion tract lengths genome-wide, without affecting crossover formation. Contrary to expectations, Mer3 helicase activity, proposed to extend the displacement loop (D-loop) recombination intermediate, does not influence the length of gene conversion events, revealing non-catalytical roles of Mer3. In addition, both purified Mer3 and MutLβ preferentially recognize D-loops, providing a mechanism for limiting gene conversion in vivo. These findings show that MutLβ is an integral part of a new regulatory step of meiotic recombination, which has implications to prevent rapid allele fixation and hotspot erosion in populations.

Remarkably Long-Tract Gene Conversion Induced by Fragile Site Instability in Saccharomyces cerevisiae.

Replication stress causes breaks at chromosomal locations called common fragile sites. Deletions causing loss of heterozygosity (LOH) in human tumors are strongly correlated with common fragile sites, but the role of gene conversion in LOH at fragile sites in tumors is less well studied. Here, we investigated gene conversion stimulated by instability at fragile site FS2 in the yeast Saccharomyces cerevisiae In our screening system, mitotic LOH events near FS2 are identified by production of red/white sectored colonies. We analyzed single nucleotide polymorphisms between homologs to determine the cause and extent of LOH. Instability at FS2 increases gene conversion 48- to 62-fold, and conversions unassociated with crossover represent 6-7% of LOH events. Gene conversion can result from repair of mismatches in heteroduplex DNA during synthesis-dependent strand annealing (SDSA), double-strand break repair (DSBR), and from break-induced replication (BIR) that switches templates [double BIR (dBIR)]. It has been proposed that SDSA and DSBR typically result in shorter gene-conversion tracts than dBIR. In cells under replication stress, we found that bidirectional tracts at FS2 have a median length of 40.8 kb and a wide distribution of lengths; most of these tracts are not crossover-associated. Tracts that begin at the fragile site FS2 and extend only distally are significantly shorter. The high abundance and long length of noncrossover, bidirectional gene-conversion tracts suggests that dBIR is a prominent mechanism for repair of lesions at FS2, thus this mechanism is likely to be a driver of common fragile site-stimulated LOH in human tumors.