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

在减数分裂中，两个连续的细胞分裂导致二倍体细胞产生单倍体配子。在第一次减数分裂中，来自父亲和母亲的同源染色体必须分离。防止分离错误的机制要求同源物在第一次减数分裂前期通过重组形成的交叉建立物理联系。减数分裂重组开始于DNA断裂的程序化形成。200-400次/细胞（小鼠）。这些断裂产生的单链DNA末端侵入同源DNA，导致同源物配对。大多数DNA断裂是通过非交叉重组修复的，这种重组只产生局部基因转换，而不是交叉，而且只有少数（在小鼠中通常是一两个）断裂在每个同源对上转化为交叉。每条染色体上的多个DNA链入侵事件中至少有一个交叉形式是至关重要的，但人们对交叉或非交叉修复之间的选择是如何做出的，以及如何控制不同的重组途径以确保及时的DNA修复，了解甚少。我们在筛选中发现MES19是一种在交叉位点积累的减数分裂特异性蛋白。我们发现MES19在交叉形成和非交叉重组中间物的及时修复中都是至关重要的。我们的分析表明，MES19是指定重组中间体作为交叉的关键参与者。我们将验证这一假设，并定位MES19和已知的前交叉蛋白在减数分裂交叉形成和DNA断裂修复中的相对功能。我们的方法将包括MES19缺陷小鼠和各种减数分裂重组突变模型（如he10， cnt1, Mlh3）的完整表型和遗传分析，以建立MES19的功能和上位性关系。特别是，减数分裂重组将通过重组标记的细胞学分析和四分体分析在我们的小鼠模型中进行分析。后者是一种非常强大的前沿方法，可以直接在DNA序列水平上分析重组结果，从而可以得出关于重组特征的准确结论。在此基础上，我们将利用生物化学和酵母两种杂交方法研究MES19的蛋白质相互作用。这些共同的努力通过揭示MES19在重组中的功能，为揭示交叉形成和DNA断裂修复在哺乳动物减数分裂中的机制提供了重要的新见解。鉴于正确执行减数分裂重组对人类生育、基因组健康和预防非整倍体至关重要，因此提出的工作具有重要的生殖相关医学意义。

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.