Genetic Recombination of c. Elegans

描述(由申请人提供)： 我们的长期目标是了解基因重组如何有助于染色体的忠实遗传。遗传重组对有性繁殖生物体至关重要，因为同源染色体的DNA分子之间的交叉重组事件以及由此产生的交叉，是减数分裂I分裂中适当的染色体分离所必需的。未能形成交叉会导致染色体错误分离和随之而来的非整倍体，这是人类流产和出生缺陷的主要原因之一。减数分裂交叉是通过故意诱导双链DNA断裂(DSB)完成的，随后在减数分裂特有的染色体结构的背景下，使用减数分裂特有的DSBR途径修改这些断裂来修复这些断裂。这一过程受到多层次的调控，以确保DSB在适当的时间背景下形成和修复，既避免对基因组完整性构成威胁，又保证每个染色体对将经历促进同源分离所需的预定交叉。我们正在研究促进和限制同源交叉(CoS)的形成以及调节线虫DSB形成的机制。线虫是一种简单的后生动物，特别适合在单一的实验系统中结合复杂的细胞学和遗传学方法，并已知在其中运行强大的交叉控制机制。这项拟议的工作将利用最近的进展，提供使用GFP：COSA-1在活的和固定的生殖细胞中可视化新生的减数分裂CO事件的地点，直接筛选CO干扰受损的突变，在指定的地点和/或确定的时间范围内通过实验诱导重组事件，将重组蛋白定向到定义的地点，以及捕获与CO地点相关的DNA的方法。一个主要的目标是确定CO干扰的因素和机制，并了解拮抗CO形成的机制和促进CO指定和CO位置逐渐分化的机制之间的相互作用。一个相关的目标是发现可能对CO/NCO指定结果产生偏差的特征。另一个目标是了解减数分裂过程中修复伙伴利用的动态调节基础。最后，我们将分析DSB减数分裂调控网络的架构，以了解DSB的形成是如何被调制的，以维持CoS的有利影响与其产生过程的潜在有害后果之间的平衡。

DESCRIPTION (provided by applicant): Our long term goal is to understand how genetic recombination contributes to the faithful inheritance of chromosomes. Genetic recombination is of central importance to sexually reproducing organisms, since crossover recombination events between the DNA molecules of homologous chromosomes, and the resulting chiasmata, are necessary for proper chromosome segregation at the meiosis I division. Failure to form crossovers leads to chromosome missegregation and consequent aneuploidy, one of the leading causes of miscarriages and birth defects in humans. Meiotic crossing over is accomplished by deliberate induction of double-strand DNA breaks (DSBs), followed by repair of these breaks using meiosis-specific modifications of DSBR pathways in the context of meiosis-specific chromosome architecture. This process is subject to multiple levels of regulation to ensure that DSBs are formed and repaired in an appropriate temporal context, both to avoid posing a threat to genome integrity and to guarantee that each chromosome pair will undergo the obligate crossover required to promote homolog segregation. We are investigating the mechanisms that promote and limit the formation of interhomolog crossovers (COs) and that regulate DSB formation in the nematode C. elegans, a simple metazoan organism that is especially amenable to combining sophisticated cytological and genetic approaches in a single experimental system, and in which robust crossover control mechanisms are known to operate. The proposed work will exploit recent advances that provide the means to visualize the sites of nascent meiotic CO events in both live and fixed germ cells using GFP:COSA-1, to screen directly for mutants with impaired CO interference, to experimentally induce recombination events at defined sites and/or defined time frames, to target recombination proteins to defined sites, and to capture DNA associated with CO sites. One major goal is to identify factors and mechanisms that contribute to CO interference, and to understand the interplay between mechanisms that antagonize CO formation and mechanisms that promote CO designation and progressive differentiation of CO sites. A related goal is to uncover features that may bias the outcome of CO/NCO designation. Another goal is to understand the basis of dynamic regulation of repair partner utilization during meiotic progression. Finally, we will analyze the architecture of the meiotic DSB regulation network to understand how DSB formation is modulated to maintain a balance between the beneficial effects of COs and the potential harmful consequences of the process by which they are generated.