Recombination pathway and partner choice during C. elegans meiosis

描述(申请人提供)：染色体之间的重组需要产生遗传变异，通过修复双链DNA断裂(DSB)来维持基因组的完整性，并确保减数分裂过程中适当的染色体分离，减数分裂是二倍体生物产生单倍体配子(如精子和卵子)的专门细胞分裂程序。重组事件中的干扰会损害这些基本的细胞功能，最终导致癌症、生育问题或出生缺陷。减数分裂重组是由DSB启动的，DSB使用减数分裂特有的机制修复，这种机制有利于利用同源染色体(而不是姐妹染色单体)作为重组伙伴，并促进DSB修复(DSBR)过程的交叉(而不是非交叉)结果。同源染色体之间的交叉重组需要建立临时的物理连接，以促进减数分裂过程中适当的染色体分离。为了促进这些重组途径和在同源染色体之间产生交叉所需的伴侣偏好，生殖细胞在减数分裂前期开始时进行专门的DSBR计划。在减数分裂后期，减数分裂细胞经历DSBR要求的第二次切换，这被认为是恢复利用有丝分裂细胞的典型修复偏好(例如，倾向于姐妹染色单体作为 修复合作伙伴)。这一提议的转换可以在减数分裂之前修复任何剩余的DSB，从而保持基因组的完整性。利用线虫的实验系统，拟议的研究将解决如何在减数分裂过程中使用不同的重组途径和伙伴来促进同源物之间的交叉形成，并在减数分裂之前修复任何剩余的DSB。为了评估这些途径和伴侣选择的时间和机制特征，我将扩大我们最近发表的检测系统的范围，开发一种检测和区分在特定部位诱导的DSB的不同修复产物的检测方法。通过这项测试，我将直接测试减数分裂前期进程中伴侣偏好的假设开关，并确定染色体位置对特定修复结果和伴侣选择的影响。此外，利用各种遗传学和细胞学技术，我将评估已知的减数分裂的作用 促进特定减数分裂修复结果的蛋白质。此外，我将利用我产生的两种新试剂来评估活虫减数分裂DSBR早期阶段的动力学，建立减数分裂染色体结构在调节DSB形成和修复中的作用，并确定特定修复伙伴选择和DSBR早期阶段动力学类别之间的关系。总体而言，这些研究将揭示重组途径和伴侣偏好如何确保染色体形成染色体分离所需的连接，并修复DSB以保持基因组的完整性。

DESCRIPTION (provided by applicant): Recombination between chromosomes is required to generate genetic variation, maintain genome integrity through the repair of double strand DNA breaks (DSBs), and ensure proper chromosome segregation during meiosis, the specialized cell division program by which diploid organisms generate haploid gametes such as sperm and eggs. Perturbations in recombination events can compromise these basic cellular functions, ultimately leading to cancer, fertility problems, or birth defects. Meiotic recombination is initiaed by DSBs, which are repaired using meiosis-specific mechanisms that favor utilization of the homologous chromosome (instead of the sister chromatid) as the recombination partner and that promote a crossover (rather than noncrossover) outcome of the DSB repair (DSBR) process. Crossover recombination between homologous chromosomes is required to create temporary physical connections that promote proper chromosome segregation during meiosis. In order to promote these recombination pathway and partner preferences required for generating crossovers between homologous chromosomes, germ cells engage a specialized DSBR program at the onset of meiotic prophase. During late meiotic prophase, meiotic cells undergo a second switch in the DSBR requirements, which is proposed to revert to utilization of repair preferences typical of mitotically dividing cells (e.g. favoring the sister chromatid as the repair partner). This proposed switch would enable repair of any remaining DSBs prior to the meiotic divisions, thereby preserving genomic integrity. Utilizing the C. elegans experimental system, the proposed studies will address how different recombination pathways and partners are employed during meiosis to both promote crossover formation between homologs and repair any remaining DSBs prior to the meiotic divisions. To assess the temporal and mechanistic features of these pathway and partner choices, I will expand the scope of our recently published assay system by developing an assay to detect and distinguish the different repair products of DSBs induced at a defined site. With this assay, I will directly test for the hypothesized switch in partner preference during meiotic prophase progression and determine the effect of chromosomal position on specific repair outcomes and partner choices. Also, utilizing a variety of genetic and cytological techniques, I will assess the roles of known meiotic proteins in promoting specific meiotic repair outcomes. Further, I will utilize two new reagents I generated to assess dynamics of the early stages of meiotic DSBR in live worms, establish the roles of meiotic chromosome structures in regulating DSB formation and repair, and determine a relationship between specific repair partner choices and classes of early DSBR stage dynamics. Overall, these studies will reveal how recombination pathway and partner preferences ensure that chromosomes form the connections necessary for chromosome segregation and repair DSBs for maintaining genomic integrity.