Completion of DNA break repair and crossover formation in mammalian meiosis; the critical functions of a previously uncharacterised meiotic protein, MES19

哺乳动物减数分裂中DNA断裂修复和交叉形成的完成；

• 批准号: 400013308
• 负责人: Professor Dr. Attila Tóth
• 金额: --
• 依托单位: Institut für Physiologische Chemie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2018
• 资助国家: 德国
• 起止时间: 2017-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/400013308?language=en
• 关键词: Completion; DNA; break; repair; crossover

In meiosis, two consecutive cell divisions lead to generation of haploid gametes from diploid cells. During the first meiotic division, homologous chromosomes (homologues) originating from the father and mother must segregate. The mechanisms preventing segregation errors require that homologues establish physical linkages via crossovers that form by recombination during the first meiotic prophase. Meiotic recombination initiates with the programmed formation of DNA breaks (appr. 200-400 breaks/cell in mice). Single-stranded DNA ends generated by these breaks invade homologous DNA, which leads to the pairing of homologues. Most DNA breaks are repaired by non-crossover recombination, which generates only local gene-conversions but not crossovers, and only few (typically one or two in mice) breaks are turned into crossovers on each homologue pair. It is essential that at least one crossover forms from the multiple DNA strand-invasion events on each chromosome but it is poorly understood how choices between crossover or non-crossover repair are made, and how distinct recombination pathways are controlled to ensure timely DNA repair. We identified MES19 in a screen as a meiosis-specific protein that accumulates at crossover sites. We found that MES19 is crucial for both crossover formation and timely repair of non-crossover recombination intermediates. Our analysis suggests that MES19 is a key player in designating recombination intermediates as crossovers. We will test this hypothesis and position the functions of MES19 and known pro-crossover proteins relative to each other in meiotic crossover formation and DNA break repair. Our approaches will include full phenotypic and genetic analyses of Mes19-deficient mice and various meiotic recombination mutant models (e.g. Hei10, Cntd1, Mlh3) to establish functional and epistatic relationships of MES19. In particular, meiotic recombination will be assayed by both cytological analysis of recombination markers and tetrad analysis in our mouse models. The latter is a very powerful cutting edge method that directly assays recombination outcomes at DNA sequence level, and thus allows making accurate conclusions about the characteristics of recombination. Complementing these, we will address protein interactions of MES19 using biochemistry and yeast two hybrid assays. These combined efforts promise crucial new insights into mechanisms of crossover formation and DNA break repair in mammalian meiosis by revealing MES19 functions in recombination. Given that correct execution of meiotic recombination is crucial for human fertility, genome health and prevention of aneuploidies, the proposed work has important reproduction-related medical implications.

在减数分裂中，两次连续的细胞分裂导致从二倍体细胞产生单倍体配子。在第一次减数分裂期间，来自父亲和母亲的同源染色体（同系物）必须分离。防止分离错误的机制需要同源物通过在第一次减数分裂前期通过重组形成的交换建立物理连锁。减数分裂重组起始于DNA断裂的程序性形成（约1990年）。小鼠中200-400个断裂/细胞）。 由这些断裂产生的单链DNA末端侵入同源DNA，这导致同源物的配对。大多数DNA断裂是通过非交换重组修复的，这只会产生局部基因转换，而不会产生交换，只有很少的（通常在小鼠中有一个或两个）断裂会在每个同源对上转化为交换。 重要的是，每个染色体上的多个DNA链侵入事件至少形成一个交叉，但人们对如何在交叉或非交叉修复之间进行选择以及如何控制不同的重组途径以确保及时的DNA修复知之甚少。我们在筛选中将MES 19鉴定为在交叉位点积累的减数分裂特异性蛋白。我们发现，MES 19是至关重要的交叉形成和及时修复非交叉重组中间体。我们的分析表明，MES 19是一个关键的球员在指定重组中间体作为交叉。我们将测试这一假设，并定位MES 19和已知的亲交换蛋白相对于彼此在减数分裂交换形成和DNA断裂修复的功能。我们的方法将包括对Mes 19缺陷小鼠和各种减数分裂重组突变模型（例如Hei 10，Cntd 1，Mlh 3）进行完整的表型和遗传分析，以建立MES 19的功能和上位关系。特别地，在我们的小鼠模型中，将通过重组标记的细胞学分析和四分体分析来测定减数分裂重组。后者是一种非常强大的前沿方法，可以直接在DNA序列水平上测定重组结果，从而可以对重组的特征做出准确的结论。补充这些，我们将使用生物化学和酵母双杂交测定解决MES 19的蛋白质相互作用。这些联合努力通过揭示MES 19在重组中的功能，有望对哺乳动物减数分裂中的交叉形成和DNA断裂修复机制产生重要的新见解。鉴于正确执行减数分裂重组对人类生育力，基因组健康和预防非整倍性至关重要，拟议的工作具有重要的生殖相关医学意义。