NBS1 Is Required For DNA Double-strand Break Repair In Meiotic Prophase

DNA double-strand break(DSB)is one of the most serious types of DNA damage.In order to deal with DSBs effectively,two classical repair pathways have been evolved in somatic cells: Homologous recombination(HR)and Non-homologous end joining(NHEJ).HR needs homologous sequences as templates for accurate repair,while NHEJ directly connects the ends of broken DNA and this approach usually causes genomic mutations.In somatic cells,once the DSBs are formed,the MRE11-RAD50-NBS1(MRN)complex will be recruited to the DSB sites.Then MRE11 uses its endonuclease and 3'-5' exonuclease activities to form a 3' single strand DNA at the end of DSBs.Subsequently,RPA2 and RAD51 mediate the invasion of single strand DNA to search for homologous template and complete HR repair.Therefore,MRN complex-dependent DSB end resection becomes the prerequisite for HR repair.In spermatocytes,numerous programed DSBs are generated by SPO11 during meiotic prophase,which are exclusively repaired by homologous recombination to promote obligate crossover between homologous chromosomes.Interestingly,These DSB ends produced during meiotic prophase are all linked with SPO11,forming SPO11-linked DSBs specifically.Although the roles of MRN homologs in meiotic DSB repair in lower organisms have been discovered,its role in mammalian meiosis remains unclear.In this study,Vasa-Cre,Stra8-GFPCre and Blimp1-Cre transgenic mice were used to specifically delete Nbs1 in germ cells,and we have demonstrated that MRN complex is critical for repairing SPO11-linked DSBs formed during meiosis in mice.In spermatocytes,conditional knockout of NBS1 results in dramatic reduction of DSB end resection and causes defective HR repair in meiotic prophase.NBS1 loss severely disrupts chromosome synapsis,generates abnormal chromosome structures,and eventually leads to zygotene arrest and male infertility in mice.In somatic cells,the recruitment of NBS1 to DSB sites is dependent on MDC1 and then NBS1 can induce ATM activation.However,in spermatocytes,we find that the recruitment of NBS1 to SPO11-linked DSB sites is independent of MDC1 and requires the interaction between NBS1-FHA domain and other unknown phosphorylated protein(s).In addition,we also find that ATM activation in NBS1 knockout spermatocytes is unaffected,which is also inconsistent with the damage-repair pathway in somatic cells.Collectively,these results suggest that there may be a specific DNA damage repair pathway in spermatocytes which is different from that in somatic cells.In addition,we also used Vasa-Cre mice to specifically delete Mre11 in germ cells.We find that the spermatogonia stem cells in the testes of MRE11 conditional knockout mice are absent rapidly and this eventually leads to male infertility.Therefore,we get the conclusion that MRE11 not only plays important roles in dealing with SPO11-linked DSBs in spermatocytes,but also has vital functions in the development of spermatogonia stem cells.Together,our studies not only reveal the significance of MRN complex in repairing SPO11-linked DSBs during meiosis,but also find that MRE11 and NBS1 have different biological functions in the development of spermatogonia stem cells.More importantly,we may find a new mechanism of DNA damage repair during meiosis,which provides a theoretical basis for studying the roles of other DNA damage repair proteins during mammalian reproductive development.