Processing and Visualization of Protec-Seq Data for the Generation of Calibrated, Genome-Wide Double Double-Strand Break (dDSB) Maps.

This chapter describes the computational pipeline for the processing and visualization of Protec-Seq data, a method for purification and genome-wide mapping of double-stranded DNA protected by a specific protein at both ends. In the published case, the protein of choice was Saccharomyces cerevisiae Spo11, a conserved topoisomerase-like enzyme that makes meiotic double-strand breaks (DSBs) to initiate homologous recombination, ensuring proper segregation of homologous chromosomes and fertility. The isolated DNA molecules were thus termed double DSB (dDSB) fragments and were found to represent 34 to several hundred base-pair long segments that are generated by Spo11 and are enriched at DSB hotspots, which are sites of topological stress. In order to allow quantitative comparisons between dDSB profiles across experiments, we implemented calibrated chromatin immunoprecipitation sequencing (ChIP-Seq) using the meiosis-competent yeast species Saccharomyces kudriavzevii as calibration strain. Here, we provide a detailed description of the computational methods for processing, analyzing, and visualizing Protec-Seq data, comprising the download of the raw data, the calibrated genome-wide alignments, and the scripted creation of either arc plots or Hi-C-style heatmaps for the illustration of chromosomal regions of interest. The workflow is based on Linux shell scripts (including wrappers for publicly available, open-source software) as well as R scripts and is highly customizable through its modular structure.

Identification and expression analysis of dmc1 in Chinese soft-shell turtle (Pelodiscus sinensis) during gametogenesis.

DNA meiotic recombinase 1 (disrupted meiotic cDNA, Dmc1) protein is homologous to the Escherichia coli RecA protein, was first identified in Saccharomyces cerevisiae. This gene has been well studied as an essential role in meiosis in many species. However, studies on the dmc1 gene in reptiles are limited. In this study, a cDNA fragment of 1,111 bp was obtained from the gonadal tissues of the Chinese soft-shell turtle via RT-PCR, containing a 60 bp 3' UTR, a 22 bp 5' UTR, and an ORF of 1,029 bp encoding 342 amino acids, named Psdmc1. Multiple sequence alignments showed that the deduced protein has high similarity (>95 %) to tetrapod Dmc1 proteins, while being slightly lower (86-88 %) to fish species.Phylogenetic tree analysis showed that PsDmc1 was clustered with the other turtles' Dmc1 and close to the reptiles', but far away from the teleost's. RT-PCR and RT-qPCR analyses showed that the Psdmc1 gene was specifically expressed in the gonads, and much higher in testis than the ovary, especially highest in one year-old testis. In situ hybridization results showed that the Psdmc1 was mainly expressed in the perinuclear cytoplasm of primary and secondary spermatocytes, weakly in spermatogonia of the testes. These results indicated that dmc1 would be majorly involved in the developing testis, and play an essential role in the germ cells' meiosis. The findings of this study will provide a basis for further investigations on the mechanisms behind the germ cells' development and differentiation in Chinese soft-shell turtles, even in the reptiles.

Diversity in Recombination Hotspot Characteristics and Gene Structure Shape Fine-Scale Recombination Patterns in Plant Genomes.

During the meiosis of many eukaryote species, crossovers tend to occur within narrow regions called recombination hotspots. In plants, it is generally thought that gene regulatory sequences, especially promoters and 5' to 3' untranslated regions, are enriched in hotspots, but this has been characterized in a handful of species only. We also lack a clear description of fine-scale variation in recombination rates within genic regions and little is known about hotspot position and intensity in plants. To address this question, we constructed fine-scale recombination maps from genetic polymorphism data and inferred recombination hotspots in 11 plant species. We detected gradients of recombination in genic regions in most species, yet gradients varied in intensity and shape depending on specific hotspot locations and gene structure. To further characterize recombination gradients, we decomposed them according to gene structure by rank and number of exons. We generalized the previously observed pattern that recombination hotspots are organized around the boundaries of coding sequences, especially 5' promoters. However, our results also provided new insight into the relative importance of the 3' end of genes in some species and the possible location of hotspots away from genic regions in some species. Variation among species seemed driven more by hotspot location among and within genes than by differences in size or intensity among species. Our results shed light on the variation in recombination rates at a very fine scale, revealing the diversity and complexity of genic recombination gradients emerging from the interaction between hotspot location and gene structure.

The non-canonical bivalent gene Wfdc15a controls spermatogenic protease and immune homeostasis.

Male infertility can be caused by chromosomal abnormalities, mutations and epigenetic defects. Epigenetic modifiers pre-program hundreds of spermatogenic genes in spermatogonial stem cells (SSCs) for expression later in spermatids, but it remains mostly unclear whether and how those genes are involved in fertility. Here, we report that Wfdc15a, a WFDC family protease inhibitor pre-programmed by KMT2B, is essential for spermatogenesis. We found that Wfdc15a is a non-canonical bivalent gene carrying both H3K4me3 and facultative H3K9me3 in SSCs, but is later activated along with the loss of H3K9me3 and acquisition of H3K27ac during meiosis. We show that WFDC15A deficiency causes defective spermiogenesis at the beginning of spermatid elongation. Notably, depletion of WFDC15A causes substantial disturbance of the testicular protease-antiprotease network and leads to an orchitis-like inflammatory response associated with TNFα expression in round spermatids. Together, our results reveal a unique epigenetic program regulating innate immunity crucial for fertility.

Rejuvenation of aged oocyte through exposure to young follicular microenvironment.

Reproductive aging is a major cause of fertility decline, attributed to decreased oocyte quantity and developmental potential. A possible cause is aging of the surrounding follicular somatic cells that support oocyte growth and development by providing nutrients and regulatory factors. Here, by creating chimeric follicles, whereby an oocyte from one follicle was transplanted into and cultured within another follicle whose native oocyte was removed, we show that young oocytes cultured in aged follicles exhibited impeded meiotic maturation and developmental potential, whereas aged oocytes cultured within young follicles were significantly improved in rates of maturation, blastocyst formation and live birth after in vitro fertilization and embryo implantation. This rejuvenation of aged oocytes was associated with enhanced interaction with somatic cells, transcriptomic and metabolomic remodeling, improved mitochondrial function and higher fidelity of meiotic chromosome segregation. These findings provide the basis for a future follicular somatic cell-based therapy to treat female infertility.

Conditional ablation of DIS3L2 ribonuclease in pre-meiotic germ cells causes defective spermatogenesis and infertility in male mice.

Rationale: Spermatogenesis is a highly organized cell differentiation process in mammals, involving mitosis, meiosis, and spermiogenesis. DIS3L2, which is primarily expressed in the cytoplasm, is an RNA exosome-independent ribonuclease. In female mice, Dis3l2-deficient oocytes fail to resume meiosis, resulting in arrest at the germinal vesicle stage and complete infertility. However, the role of DIS3L2 in germ cell development in males has remained largely unexplored. Methods: We established a pre-meiotic germ cell conditional knockout mouse model and investigated the biological function of DIS3L2 in spermatogenesis and male fertility through bulk RNA-seq and scRNA-seq analyses. Results: This study unveils that conditional ablation of Dis3l2 in pre-meiotic germ cells with Stra8-Cre mice impairs spermatogonial differentiation and hinders spermatocyte meiotic progression coupled with cell apoptosis. Such conditional ablation leads to defective spermatogenesis and sterility in adults. Bulk RNA-seq analysis revealed that Dis3l2 deficiency significantly disrupted the transcriptional expression pattern of genes related to the cell cycle, spermatogonial differentiation, and meiosis in Dis3l2 conditional knockout testes. Additionally, scRNA-seq analysis indicated that absence of DIS3L2 in pre-meiotic germ cells causes disrupted RNA metabolism, downregulated expression of cell cycle genes, and aberrant expression of spermatogonial differentiation genes, impeding spermatogonial differentiation. In meiotic spermatocytes, loss of DIS3L2 results in disturbed RNA metabolism, abnormal translation, and disrupted meiotic genes that perturb meiotic progression and induce cell apoptosis, leading to subsequent failure of spermatogenesis and male infertility. Conclusions: Collectively, these findings highlight the critical role of DIS3L2 ribonuclease-mediated RNA degradation in safeguarding the correct transcriptome during spermatogonial differentiation and spermatocyte meiotic progression, thus ensuring normal spermatogenesis and male fertility.

Integrated cytological and transcriptomic analyses provide new insights into restoration of pollen viability in synthetic allotetraploid Brassica carinata.

KEY MESSAGE: Upregulation of genes involved in DNA damage repair and sperm cell differentiation leads to restoration of pollen viability in synthetic allotetraploid B. carinata after chromosome doubling. Apart from the well-known contribution of polyploidy to crop improvement, polyploids can also be induced for other purposes, such as to restore the viability of sterile hybrids. The mechanism related to viability transition between the sterile allodiploid and the fertile allotetraploid after chromosome doubling are not well understood. Here, we synthesised allodiploid B. carinata (2n = 2x = 17) and allotetraploid B. carinata (2n = 4x = 34) as models to investigate the cytological and transcriptomic differences during pollen development. The results showed that after chromosome doubling, the recovery of pollen viability in allotetraploid was mainly reflected in the stabilisation of microtubule spindle morphology, normal meiotic chromosome behaviour, and normal microspore development. Interestingly, the deposition and degradation of synthetic anther tapetum were not affected by polyploidy. Transcription analysis showed that the expression of genes related to DNA repair (DMC1, RAD51, RAD17, SPO11-2), cell cycle differentiation (CYCA1;2, CYCA2;3) and ubiquitination proteasome pathway (UBC4, PIRH2, CDC53) were positively up-regulated during pollen development of synthetic allotetraploid B. carinata. In summary, these results provide some refreshing updates about the ploidy-related restoration of pollen viability in newly synthesised allotetraploid B. carinata.

Positive Selection Drives the Evolution of the Structural Maintenance of Chromosomes (SMC) Complexes.

Structural Maintenance of Chromosomes (SMC) complexes are an evolutionary conserved protein family. In most eukaryotes, three SMC complexes have been characterized, as follows: cohesin, condensin, and SMC5/6 complexes. These complexes are involved in a plethora of functions, and defects in SMC genes can lead to an increased risk of chromosomal abnormalities, infertility, and cancer. To investigate the evolution of SMC complex genes in mammals, we analyzed their selective patterns in an extended phylogeny. Signals of positive selection were identified for condensin NCAPG, for two SMC5/6 complex genes (SMC5 and NSMCE4A), and for all cohesin genes with almost exclusive meiotic expression (RAD21L1, REC8, SMC1B, and STAG3). For the latter, evolutionary rates correlate with expression during female meiosis, and most positively selected sites fall in intrinsically disordered regions (IDRs). Our results support growing evidence that IDRs are fast evolving, and that they most likely contribute to adaptation through modulation of phase separation. We suggest that the natural selection signals identified in SMC complexes may be the result of different selective pressures: a host-pathogen arms race in the condensin and SMC5/6 complexes, and an intragenomic conflict for meiotic cohesin genes that is similar to that described for centromeres and telomeres.

BRCA2 stabilises RAD51 and DMC1 nucleoprotein filaments through a conserved interaction mode.

BRCA2 is essential for DNA repair by homologous recombination in mitosis and meiosis. It interacts with recombinases RAD51 and DMC1 to facilitate the formation of nucleoprotein filaments on resected DNA ends that catalyse recombination-mediated repair. BRCA2's BRC repeats bind and disrupt RAD51 and DMC1 filaments, whereas its PhePP motifs bind recombinases and stabilise their nucleoprotein filaments. However, the mechanism of filament stabilisation has hitherto remained unknown. Here, we report the crystal structure of a BRCA2-DMC1 complex, revealing how core interaction sites of PhePP motifs bind to recombinases. The interaction mode is conserved for RAD51 and DMC1, which selectively bind to BRCA2's two distinct PhePP motifs via subtly divergent binding pockets. PhePP motif sequences surrounding their core interaction sites protect nucleoprotein filaments from BRC-mediated disruption. Hence, we report the structural basis of how BRCA2's PhePP motifs stabilise RAD51 and DMC1 nucleoprotein filaments for their essential roles in mitotic and meiotic recombination.

FBXO47 regulates centromere pairing as key component of centromeric SCF E3 ligase in mouse spermatocytes.

Centromere pairing is crucial for synapsis in meiosis. This study delves into the Skp1-Cullin1-F-box protein (SCF) E3 ubiquitin ligase complex, specifically focusing on F-box protein 47 (FBXO47), in mouse meiosis. Here, we revealed that FBXO47 is localized at the centromere and it regulates centromere pairing cooperatively with SKP1 to ensure proper synapsis in pachynema. The absence of FBXO47 causes defective centromeres, resulting in incomplete centromere pairing, which leads to corruption of SC at centromeric ends and along chromosome axes, triggering premature dissociation of chromosomes and pachytene arrest. FBXO47 deficient pachytene spermatocytes exhibited drastically reduced SKP1 expression at centromeres and chromosomes. Additionally, FBXO47 stabilizes SKP1 by down-regulating its ubiquitination in HEK293T cells. In essence, we propose that FBXO47 collaborates with SKP1 to facilitate centromeric SCF formation in spermatocytes. In summary, we posit that the centromeric SCF E3 ligase complex regulates centromere pairing for pachynema progression in mice.

G-Protein-Coupled Receptor Kinase 2 Inhibition Induces Meiotic Arrest by Disturbing Ca2+ Release in Porcine Oocytes.

G-protein-coupled receptor kinase 2 (GRK2) interacts with Gßγ and Gαq, subunits of G-protein alpha, to regulate cell signalling. The second messenger inositol trisphosphate, produced by activated Gαq, promotes calcium release from the endoplasmic reticulum (ER) and regulates maturation-promoting factor (MPF) activity. This study aimed to investigate the role of GRK2 in MPF activity during the meiotic maturation of porcine oocytes. A specific inhibitor of GRK2 (ßi) was used in this study. The present study showed that GRK2 inhibition increased the percentage of oocyte arrest at the metaphase I (MI) stage (control: 13.84 ± 0.95%; ßi: 31.30 ± 4.18%), which resulted in the reduction of the maturation rate (control: 80.36 ± 1.94%; ßi: 65.40 ± 1.14%). The level of phospho-GRK2 decreased in the treated group, suggesting that GRK2 activity was reduced upon GRK2 inhibition. Furthermore, the addition of ßi decreased Ca2+ release from the ER. The protein levels of cyclin B and cyclin-dependent kinase 1 were higher in the treatment group than those in the control group, indicating that GRK2 inhibition prevented a decrease in MPF activity. Collectively, GRK2 inhibition induced meiotic arrest at the MI stage in porcine oocytes by preventing a decrease in MPF activity, suggesting that GRK2 is essential for oocyte meiotic maturation in pigs.

B chromosome and its non-Mendelian inheritance in Atractylodes lancea.

Supernumerary B chromosomes contribute to intraspecific karyotypic variation. B chromosomes have been detected in more than 2000 organisms; they possess unique and diverse features, including non-Mendelian inheritance. Here, we report one or more B chromosomes in the gynodioecious plant Atractylodes lancea. Among 54 A. lancea lines, 0-2 B chromosomes were detected in both hermaphroditic and female plants, with the B chromosomes appearing as DAPI-bright regions within the nuclei. Genomic in situ hybridization revealed that the B chromosomes had no conserved A chromosome DNA sequences, confirmed by fluorescence in situ hybridization probed with independently dissected B chromosomes. In male meiosis, the B chromosome did not pair with an A chromosome and was therefore eliminated; accordingly, only 20.1% and 18.6% of these univalent B chromosomes remained at the end of meiosis for the 1B lines of KY17-148 and KY17-118, respectively. However, we also found that B chromosomes were transmitted from male parents in 40.8%-44.2% and 47.2% of the next generation; although these transmission rates from male parents were not essentially different from Mendelian inheritance (0.5), the transmission of gametes carrying B chromosomes increased through fertilization or seed development. B chromosomes were transmitted from three of four 1B female parents to 64.3%-92.6% of the next generation, suggesting B chromosome accumulation. We propose that the B chromosome of A. lancea has a specific sequence and persists via non-Mendelian inheritance from female parents. Overall, A. lancea, with its unique characteristics, is a promising model for understanding the structure, evolution, and mechanism of non-Mendelian inheritance of B chromosomes.

Satellite DNAs and the evolution of the multiple X1X2Y sex chromosomes in the wolf fish Hoplias malabaricus (Teleostei; Characiformes).

Multiple sex chromosomes usually arise from chromosomal rearrangements which involve ancestral sex chromosomes. There is a fundamental condition to be met for their long-term fixation: the meiosis must function, leading to the stability of the emerged system, mainly concerning the segregation of the sex multivalent. Here, we sought to analyze the degree of differentiation and meiotic pairing properties in the selected fish multiple sex chromosome system present in the wolf-fish Hoplias malabaricus (HMA). This species complex encompasses seven known karyotype forms (karyomorphs) where the karyomorph C (HMA-C) exhibits a nascent XY sex chromosomes from which the multiple X1X2Y system evolved in karyomorph HMA-D via a Y-autosome fusion. We combined genomic and cytogenetic approaches to analyze the satellite DNA (satDNA) content in the genome of HMA-D karyomorph and to investigate its potential contribution to X1X2Y sex chromosome differentiation. We revealed 56 satDNA monomers of which the majority was AT-rich and with repeat units longer than 100 bp. Seven out of 18 satDNA families chosen for chromosomal mapping by fluorescence in situ hybridization (FISH) formed detectable accumulation in at least one of the three sex chromosomes (X1, X2 and neo-Y). Nine satDNA monomers showed only two hybridization signals limited to HMA-D autosomes, and the two remaining ones provided no visible FISH signals. Out of seven satDNAs located on the HMA-D sex chromosomes, five mapped also to XY chromosomes of HMA-C. We showed that after the autosome-Y fusion event, the neo-Y chromosome has not substantially accumulated or eliminated satDNA sequences except for minor changes in the centromere-proximal region. Finally, based on the obtained FISHpatterns, we speculate on the possible contribution of satDNA to sex trivalent pairing and segregation.

A partial deletion within the meiosis-specific sporulation domain SPO22 of Tex11 is not associated with infertility in mice.

Azoospermia (the complete absence of spermatozoa in the semen) is a common cause of male infertility. The etiology of azoospermia is poorly understood. Whole-genome analysis of azoospermic men has identified a number of candidate genes, such as the X-linked testis-expressed 11 (TEX11) gene. Using a comparative genomic hybridization array, an exonic deletion (exons 10-12) of TEX11 had previously been identified in two non-apparent azoospermic patients. However, the putative impact of this genetic alteration on spermatogenesis and the azoospermia phenotype had not been validated functionally. We therefore used a CRISPR/Cas9 system to generate a mouse model (Tex11Ex9-11del/Y) with a partial TEX11 deletion that mimicked the human mutation. Surprisingly, the mutant male Tex11Ex9-11del/Y mice were fertile. The sperm concentration, motility, and morphology were normal. Similarly, the mutant mouse line's testis transcriptome was normal, and the expression of spermatogenesis genes was not altered. These results suggest that the mouse equivalent of the partial deletion observed in two infertile male with azoospermia has no impact on spermatogenesis or fertility in mice, at least of a FVB/N genetic background and until 10 months of age. Mimicking a human mutation does not necessarily lead to the same human phenotype in mice, highlighting significant differences species.

The role of DNA topoisomerase 1α (AtTOP1α) in regulating arabidopsis meiotic recombination and chromosome segregation.

Meiosis is a critical process in sexual reproduction, and errors during this cell division can significantly impact fertility. Successful meiosis relies on the coordinated action of numerous genes involved in DNA replication, strand breaks, and subsequent rejoining. DNA topoisomerase enzymes play a vital role by regulating DNA topology, alleviating tension during replication and transcription. To elucidate the specific function of DNA topoisomerase 1α ( A t T O P 1 α ) in male reproductive development of Arabidopsis thaliana, we investigated meiotic cell division in Arabidopsis flower buds. Combining cytological and biochemical techniques, we aimed to reveal the novel contribution of A t T O P 1 α to meiosis. Our results demonstrate that the absence of A t T O P 1 α leads to aberrant chromatin behavior during meiotic division. Specifically, the top1α1 mutant displayed altered heterochromatin distribution and clustered centromere signals at early meiotic stages. Additionally, this mutant exhibited disruptions in the distribution of 45s rDNA signals and a reduced frequency of chiasma formation during metaphase I, a crucial stage for genetic exchange. Furthermore, the atm-2×top1α1 double mutant displayed even more severe meiotic defects, including incomplete synapsis, DNA fragmentation, and the presence of polyads. These observations collectively suggest that A t T O P 1 α plays a critical role in ensuring accurate meiotic progression, promoting homologous chromosome crossover formation, and potentially functioning in a shared DNA repair pathway with ATAXIA TELANGIECTASIA MUTATED (ATM) in Arabidopsis microspore mother cells.

Yeast EndoG prevents genome instability by degrading extranuclear DNA species.

In metazoans mitochondrial DNA (mtDNA) or retrotransposon cDNA released to cytoplasm are degraded by nucleases to prevent sterile inflammation. It remains unknown whether degradation of these DNA also prevents nuclear genome instability. We used an amplicon sequencing-based method in yeast enabling analysis of millions of DSB repair products. In non-dividing stationary phase cells, Pol4-mediated non-homologous end-joining increases, resulting in frequent insertions of 1-3 nucleotides, and insertions of mtDNA (NUMTs) or retrotransposon cDNA. Yeast EndoG (Nuc1) nuclease limits insertion of cDNA and transfer of very long mtDNA ( >10 kb) to the nucleus, where it forms unstable circles, while promoting the formation of short NUMTs (~45-200 bp). Nuc1 also regulates transfer of extranuclear DNA to nucleus in aging or meiosis. We propose that Nuc1 preserves genome stability by degrading retrotransposon cDNA and long mtDNA, while short NUMTs originate from incompletely degraded mtDNA. This work suggests that nucleases eliminating extranuclear DNA preserve genome stability.

Premeiotic endoreplication is the mechanism of obligate parthenogenesis in rock lizards of the genus Darevskia.

Among vertebrates, obligate parthenogenesis occurs exclusively in squamate reptiles. Premeiotic endoreplication in a small subset of developing oocytes has been documented as the mechanism of production of unreduced eggs in minutely explored obligate parthenogenetic lineages, namely in teiids and geckos. The situation in the lacertid genus Darevskia has been discussed for decades. Certain observations suggested that the ploidy level is restored during egg formation through a fusion of egg and polar body nuclei in Darevskia unisexualis and D. armeniaca. In this study, we re-evaluated the fusion hypothesis by studying diplotene chromosomes in adult females of sexual species D. raddei nairensis and obligate parthenogens D. armeniaca, D. dahli and D. unisexualis. We revealed 19 bivalents in the sexual species and 38 bivalents in the diploid obligate parthenogens, which uncovers premeiotic endoreplication as the mechanism of the production of non-reduced eggs in parthenogenetic females. The earlier contradicting reports can likely be attributed to the difficulty in identifying mispairing of chromosomes in pachytene, and the fact that in parthenogenetic reptiles relying on premeiotic endoreplication only a small subset of developing oocytes undergo genome doubling and overcome the pachytene checkpoint. This study highlights co-option of premeiotic endoreplication for escape from sexual reproduction in all independent hybrid origins of obligate parthenogenesis in vertebrates studied to date.

PRSS50-mediated inhibition of MKP3/ERK signaling is crucial for meiotic progression and sperm quality.

Serine protease 50 (PRSS50/TSP50) is highly expressed in spermatocytes. Our study investigated its role in testicular development and spermatogenesis. Initially, PRSS50 knockdown was observed to impair DNA synthesis in spermatocytes. To further explore this, we generated PRSS50 knockout ( Prss50 -/- ) mice ( Mus musculus), which exhibited abnormal spermatid nuclear compression and reduced male fertility. Furthermore, dysplastic seminiferous tubules and decreased sex hormones were observed in 4-week-old Prss50 -/- mice, accompanied by meiotic progression defects and increased apoptosis of spermatogenic cells. Mechanistic analysis indicated that PRSS50 deletion resulted in increased phosphorylation of extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) and elevated levels of MAP kinase phosphatase 3 (MKP3), a specific ERK antagonist, potentially accounting for testicular dysplasia in adolescent Prss50 -/- mice. Taken together, these findings suggest that PRSS50 plays an important role in testicular development and spermatogenesis, with the MKP3/ERK signaling pathway playing a significant role in this process.

Irc20 modulates LOH frequency and distribution in S. cerevisiae.

Loss of Heterozygosity (LOH) due to mitotic recombination is frequently associated with the development of various cancers (e.g. retinoblastoma). LOH is also an important source of genetic diversity, especially in organisms where meiosis is infrequent. Irc20 is a putative helicase, and E3 ubiquitin ligase involved in DNA double-strand break repair pathway. We analyzed genome-wide LOH events, gross chromosomal changes, small insertion-deletions and single nucleotide mutations in eleven S. cerevisiae mutation accumulation lines of irc20∆, which underwent 50 mitotic bottlenecks. LOH enhancement in irc20∆ was small (1.6 fold), but statistically significant as compared to the wild type. Short (≤ 1 kb) and long (> 10 kb) LOH tracts were significantly enhanced in irc20∆. Both interstitial and terminal LOH events were also significantly enhanced in irc20∆ compared to the wild type. LOH events in irc20∆ were more telomere proximal and away from centromeres compared to the wild type. Gross chromosomal changes, single nucleotide mutations and in-dels were comparable between irc20∆ and wild type. Locus based and genome-wide analysis of meiotic recombination showed that meiotic crossover frequencies are not altered in irc20∆. These results suggest Irc20 primarily regulates mitotic recombination and does not affect meiotic crossovers. Our results suggest that the IRC20 gene is important for regulating LOH frequency and distribution.