COM-1 promotes homologous recombination during Caenorhabditis elegans meiosis by antagonizing Ku-mediated non-homologous end joining.
ABSTRACT: Successful completion of meiosis requires the induction and faithful repair of DNA double-strand breaks (DSBs). DSBs can be repaired via homologous recombination (HR) or non-homologous end joining (NHEJ), yet only repair via HR can generate the interhomolog crossovers (COs) needed for meiotic chromosome segregation. Here we identify COM-1, the homolog of CtIP/Sae2/Ctp1, as a crucial regulator of DSB repair pathway choice during Caenorhabditis elegans gametogenesis. COM-1-deficient germ cells repair meiotic DSBs via the error-prone pathway NHEJ, resulting in a lack of COs, extensive chromosomal aggregation, and near-complete embryonic lethality. In contrast to its yeast counterparts, COM-1 is not required for Spo11 removal and initiation of meiotic DSB repair, but instead promotes meiotic recombination by counteracting the NHEJ complex Ku. In fact, animals defective for both COM-1 and Ku are viable and proficient in CO formation. Further genetic dissection revealed that COM-1 acts parallel to the nuclease EXO-1 to promote interhomolog HR at early pachytene stage of meiotic prophase and thereby safeguards timely CO formation. Both of these nucleases, however, are dispensable for RAD-51 recruitment at late pachytene stage, when homolog-independent repair pathways predominate, suggesting further redundancy and/or temporal regulation of DNA end resection during meiotic prophase. Collectively, our results uncover the potentially lethal properties of NHEJ during meiosis and identify a critical role for COM-1 in NHEJ inhibition and CO assurance in germ cells.
Project description:Chromosome inheritance during sexual reproduction relies on deliberate induction of double-strand DNA breaks (DSBs) and repair of a subset of these breaks as interhomolog crossovers (COs). Here we provide a direct demonstration, based on our analysis of rad-50 mutants, that the meiotic program in Caenorhabditis elegans involves both acquisition and loss of a specialized mode of double-strand break repair (DSBR). In premeiotic germ cells, RAD-50 is not required to load strand-exchange protein RAD-51 at sites of spontaneous or ionizing radiation (IR)-induced DSBs. A specialized meiotic DSBR mode is engaged at the onset of meiotic prophase, coincident with assembly of meiotic chromosome axis structures. This meiotic DSBR mode is characterized both by dependence on RAD-50 for rapid accumulation of RAD-51 at DSB sites and by competence for converting DSBs into interhomolog COs. At the mid-pachytene to late pachytene transition, germ cells undergo an abrupt release from the meiotic DSBR mode, characterized by reversion to RAD-50-independent loading of RAD-51 and loss of competence to convert DSBs into interhomolog COs. This transition in DSBR mode is dependent on MAP kinase-triggered prophase progression and coincides temporally with a major remodeling of chromosome architecture. We propose that at least two developmentally programmed switches in DSBR mode, likely conferred by changes in chromosome architecture, operate in the C. elegans germ line to allow formation of meiotic crossovers without jeopardizing genomic integrity. Our data further suggest that meiotic cohesin component REC-8 may play a role in limiting the activity of SPO-11 in generating meiotic DSBs and that RAD-50 may function in counteracting this inhibition.
Project description:Meiotic recombination is initiated by DNA double-strand breaks (DSBs). Most DSBs are converted into nonreciprocal exchanges (gene conversions) or crossovers (COs) between sister chromatids. Only a minority of DSBs are processed toward interhomolog COs, the precursors of the chiasmata that connect homologous chromosomes. Dmc1, the meiosis-specific paralog of the universal recombination protein Rad51, is required for interhomolog COs; in its absence, univalents are primarily formed. Here, we report a ciliate-specific novel meiotic gene, BIME2, which also promotes interhomolog crossing over. In the bime2? mutant, DSBs are formed and repaired normally, but bivalent formation is strongly reduced. Bime2 protein forms foci on chromatin during meiotic prophase, and chromatin localization of Bime2 and Dmc1 is largely interdependent. Bime2 distantly resembles budding yeast Rdh54/Tid1 and the vertebrate Rad54B helicases and may have similar functions in promoting or stabilizing Dmc1 nucleoprotein filaments.
Project description:Repair of double-strand DNA breaks (DSBs) by the homologous recombination (HR) pathway results in crossovers (COs) required for a successful first meiotic division. Mre11 is one member of the MRX/N (Mre11, Rad50, and Xrs2/Nbs1) complex required for meiotic DSB formation and for resection in Saccharomyces cerevisiae. In Caenorhabditis elegans, evidence for the MRX/N role in DSB resection is limited. We report the first separation-of-function allele, mre-11(iow1) in C. elegans, which is specifically defective in meiotic DSB resection but not in formation. The mre-11(iow1) mutants displayed chromosomal fragmentation and aggregation in late prophase I. Recombination intermediates and crossover formation was greatly reduced in mre-11(iow1) mutants. Irradiation-induced DSBs during meiosis failed to be repaired from early to middle prophase I in mre-11(iow1) mutants. In the absence of a functional HR, our data suggest that some DSBs in mre-11(iow1) mutants are repaired by the nonhomologous end joining (NHEJ) pathway, as removing NHEJ partially suppressed the meiotic defects shown by mre-11(iow1). In the absence of NHEJ and a functional MRX/N, meiotic DSBs are channeled to EXO-1-dependent HR repair. Overall, our analysis supports a role for MRE-11 in the resection of DSBs in middle meiotic prophase I and in blocking NHEJ.
Project description:During meiosis, programmed double-strand breaks (DSBs) are repaired via recombination pathways that are required for faithful chromosomal segregation and genetic diversity. In meiotic progression, the non-homologous end joining (NHEJ) pathway is suppressed and instead meiotic recombination initiated by nucleolytic resection of DSB ends is the major pathway employed. This requires diverse recombinase proteins and regulatory factors involved in the formation of crossovers (COs) and non-crossovers (NCOs). In mitosis, spontaneous DSBs occurring at the G1 phase are predominantly repaired via NHEJ, mediating the joining of DNA ends. The Ku complex binds to these DSB ends, inhibiting additional DSB resection and mediating end joining with Dnl4, Lif1, and Nej1, which join the Ku complex and DSB ends. Here, we report the role of the Ku complex in DSB repair using a physical analysis of recombination in Saccharomyces cerevisiae during meiosis. We found that the Ku complex is not essential for meiotic progression, DSB formation, joint molecule formation, or CO/NCO formation during normal meiosis. Surprisingly, in the absence of the Ku complex and functional Mre11-Rad50-Xrs2 (MRX) complex, a large portion of meiotic DSBs was repaired via the recombination pathway to form COs and NCOs. Our data suggested that Ku complex prevents meiotic recombination in the elimination of MRX activity. [BMB Reports 2019; 52(10): 607-612].
Project description:Faithful inheritance of genetic information through sexual reproduction relies on the formation of crossovers between homologous chromosomes during meiosis, which, in turn, relies on the formation and repair of numerous double-strand breaks (DSBs). As DSBs pose a potential threat to the genome, mechanisms that ensure timely and error-free DSB repair are crucial for successful meiosis. Here, we identify NBS-1, the Caenorhabditis elegans ortholog of the NBS1 (mutated in Nijmegen Breakage Syndrome) subunit of the conserved MRE11-RAD50-NBS1/Xrs2 (MRN) complex, as a key mediator of DSB repair via homologous recombination (HR) during meiosis. Loss of nbs-1 leads to severely reduced loading of recombinase RAD-51, ssDNA binding protein RPA, and pro-crossover factor COSA-1 during meiotic prophase progression; aggregated and fragmented chromosomes at the end of meiotic prophase; and 100% progeny lethality. These phenotypes reflect a role for NBS-1 in processing of meiotic DSBs for HR that is shared with its interacting partners MRE-11-RAD-50 and COM-1 (ortholog of Com1/Sae2/CtIP). Unexpectedly, in contrast to MRE-11 and RAD-50, NBS-1 is not required for meiotic DSB formation. Meiotic defects of the nbs-1 mutant are partially suppressed by abrogation of the nonhomologous end-joining (NHEJ) pathway, indicating a role for NBS-1 in antagonizing NHEJ during meiosis. Our data further reveal that NBS-1 and COM-1 play distinct roles in promoting HR and antagonizing NHEJ. We propose a model in which different components of the MRN-C complex work together to couple meiotic DSB formation with efficient and timely engagement of HR, thereby ensuring crossover formation and restoration of genome integrity before the meiotic divisions.
Project description:During meiosis, DNA double-strand breaks (DSBs) are physiologically induced to start the recombination process and promote the formation of interhomologue crossovers (COs), which are required to ensure faithful chromosome segregation into the gametes. The timely repair of DSBs is an essential part of the meiotic programme, as accumulation of unprocessed DSBs during the pachytene stage of meiotic prophase triggers a DNA damage checkpoint response that induces apoptosis of damaged cells. We show that CO-promoting factors MSH-4, MSH-5, and ZHP-3, but not COSA-1, are required for the apoptotic response of the meiotic DNA damage checkpoint. Lack of MSH-4 or MSH-5 suppresses the apoptotic response observed in some DNA repair-defective mutants such as fcd-2 and brc-1 (orthologues of FANCD2 and BRCA1), irrespectively of the amount of DSBs present in pachytene nuclei. Although ionizing radiation fails to induce apoptosis in msh-4/5-mutant backgrounds, it induces transcriptional activation of the apoptosis-activator egl-1, which is controlled by the Caenorhabditis elegans p53 orthologue CEP-1. This finding suggests that MSH-4/5 involvement in the apoptotic response occurs downstream or independently of damage sensing and checkpoint activation. This study establishes a role for pro-CO factors MSH-4/5 and ZHP-3 in the execution of apoptosis at late meiotic prophase following the accumulation of exogenous or endogenous DNA damage.
Project description:Spermatogenesis is a complex process that generates haploid germ cells or spores and implements meiosis, a succession of two special cell divisions that are required for homologous chromosome segregation. During prophase to the first meiotic division, homologous recombination (HR) repairs Spo11-dependent DNA double-strand breaks (DSBs) in the presence of telomere movements to allow for chromosome pairing and segregation at the meiosis I division. In contrast to HR, non-homologous end joining (NHEJ), the major DSB repair mechanism during the G1 cell cycle phase, is downregulated during early meiotic prophase. At somatic mammalian telomeres, the NHEJ factor Ku70/80 inhibits HR, as does the Rap1 component of the shelterin complex. Here, we investigated the role of Ku70 and Rap1 in meiotic telomere redistribution and genome protection in spermatogenesis by studying single and double knockout mice. Ku70(-/-) mice display reduced testis size and compromised spermatogenesis, whereas meiotic telomere dynamics and chromosomal bouquet formation occurred normally in Ku70(-/-) and Ku70(-/-)Rap1(?/?) knockout spermatocytes. Elevated mid-preleptotene frequencies were associated with significantly increased DNA damage in Ku-deficient B spermatogonia, and in differentiated Sertoli cells. Significantly elevated levels of ?H2AX foci in Ku70(-/-) diplotene spermatocytes suggest compromised progression of DNA repair at a subset of DSBs. This might explain the elevated meiotic metaphase apoptosis that is present in Ku70-deficient stage XII testis tubules, indicating spindle assembly checkpoint activation. In summary, our data indicate that Ku70 is important for repairing DSBs in somatic cells and in late spermatocytes of the testis, thereby assuring the fidelity of spermatogenesis.
Project description:Homologous recombination promotes proper segregation of chromosomes during meiosis. Programmed double-strand breaks (DSBs) initiate recombination and are repaired preferentially using the homolog rather than the sister chromatid template. In yeast, activation of Mek1 kinase upholds this bias. Mek1 is also a proposed effector kinase in the recombination checkpoint that responds to aberrant DNA and/or axis structures. Elucidating a role for Mek1 in this checkpoint has been difficult, because a mek1 null mutation causes rapid repair of DSBs using a sister chromatid, thus bypassing formation of checkpoint-activating lesions. Here we analyzed a MEK1 gain-of-function allele to test if it would enhance interhomolog bias and/or the checkpoint response.When Mek1 activation was artificially maintained through glutathione S-transferase-mediated dimerization, there was an enhanced skew toward interhomolog recombination and reduction of intersister events, including multichromatid joint molecules. Increased interhomolog events were specifically repaired as noncrossovers rather than as crossovers. Ectopic Mek1 dimerization was also sufficient to impose interhomolog bias in the absence of recombination checkpoint functions, thereby uncoupling these two processes. Finally, the stringency of the checkpoint response was enhanced in mutants with weak recombination defects by blocking prophase exit in a subset of cells in which arrest is not absolute.We propose that Mek1 plays dual roles during meiotic prophase I by phosphorylating targets directly involved in the recombination checkpoint, as well as targets involved in sister chromatid recombination. We discuss how regulation of pachytene exit by Mek1 or similar kinases could influence checkpoint stringency, which may differ among species and between sexes.
Project description:During meiosis, the maintenance of genome integrity is critical for generating viable haploid gametes.<sup>1</sup> In meiotic prophase I, double-strand DNA breaks (DSBs) are induced and a subset of these DSBs are repaired as interhomolog crossovers to ensure proper chromosome segregation. DSBs not resolved as crossovers with the homolog must be repaired by other pathways to ensure genome integrity.<sup>2</sup> To determine if alternative repair templates can be engaged for meiotic DSB repair during oogenesis, we developed an assay to detect sister and/or intra-chromatid repair events at a defined DSB site during Caenorhabditis elegans meiosis. Using this assay, we directly demonstrate that the sister chromatid or the same DNA molecule can be engaged as a meiotic repair template for both crossover and noncrossover recombination, with noncrossover events being the predominant recombination outcome. We additionally find that the sister or intra-chromatid substrate is available as a recombination partner for DSBs induced throughout meiotic prophase I, including late prophase when the homolog is unavailable. Analysis of noncrossover conversion tract sequences reveals that DSBs are processed similarly throughout prophase I. We further present data indicating that the XPF-1 nuclease functions in late prophase to promote sister or intra-chromatid repair at steps of recombination following joint molecule processing. Despite its function in sister or intra-chromatid repair, we find that xpf-1 mutants do not exhibit severe defects in progeny viability following exposure to ionizing radiation. Overall, we propose that C. elegans XPF-1 may assist as an intersister or intrachromatid resolvase only in late prophase I.
Project description:Interhomolog crossovers (COs) are a prerequisite for achieving accurate chromosome segregation during meiosis [1, 2]. COs are not randomly positioned, occurring at distinct genomic intervals during meiosis in all species examined [3-10]. The role of CO position as a major determinant of accurate chromosome segregation has not been previously directly analyzed in a metazoan. Here, we use spo-11 mutants, which lack endogenous DNA double-strand breaks (DSBs), to induce a single DSB by Mos1 transposon excision at defined chromosomal locations in the C. elegans germline and show that the position of the resulting CO directly affects the formation of distinct chromosome subdomains during meiotic chromosome remodeling. CO formation in the typically CO-deprived center region of autosomes leads to premature loss of sister chromatid cohesion and chromosome missegregation, whereas COs at an off-centered position, as in wild type, can result in normal remodeling and accurate segregation. Ionizing radiation (IR)-induced DSBs lead to the same outcomes, and modeling of IR dose-response reveals that the CO-unfavorable center region encompasses up to 6% of the total chromosome length. DSBs proximal to telomeres rarely form COs, likely because of formation of unstable recombination intermediates that cannot be sustained as chiasmata until late prophase. Our work supports a model in which regulation of CO position early in meiotic prophase is required for proper designation of chromosome subdomains and normal chromosome remodeling in late meiotic prophase I, resulting in accurate chromosome segregation and providing a mechanism to prevent aneuploid gamete formation.