Recombination mechanisms of crossover and noncrossover formation in Drosophila

描述（由申请方提供）：为确保减数分裂期间染色体的准确分离，使用重组在同源染色体之间产生交叉。通过形成程序性DNA双链断裂来启动双链断裂，通过与同源染色体的相互作用来修复双链断裂，最终形成联合DNA分子。重组模型和联合分子的拟议结构主要基于酵母中重组中间体的检测、体外生物化学测定和由于重叠的基因功能而难以解释的遗传数据。基于这些数据的模型通常被认为是所有生物体的普遍事实，而实际上在酵母菌之外几乎没有可用的体内数据。在这个建议中，我描述的实验，将确定不同的重组模型的减数分裂交叉和非交叉形成的相对贡献。我将使用后生动物黑腹果蝇和一个独特的遗传分析，允许恢复自发重组事件内的异等位基因位点，然后可以进行分析，以确定异源双链DNA的安排。我还建议使用下一代测序对重组事件进行全基因组分析。果蝇的优势在于，它是唯一已知的生物体，其中已经鉴定了95%的所有交叉所需的推定的双霍利迪连接解离酶MEI-9。通过将拟议的遗传实验与MEI-9已知的生化特性相结合，我将能够对果蝇中的重组如何起作用做出强有力的结论。这些实验的优点是在后生动物生殖系的背景下进行，并且比其他后生动物系统能够以更高的分辨率测量自发重组事件。这些实验的结果将扩大重组机制的知识，以模型系统以外的真菌和直接适用于人类生育问题和癌症。

DESCRIPTION (provided by applicant): To ensure the accurate segregation of chromosomes during meiosis, recombination is used to create a crossover between homologous chromosomes. Recombination is initiated by the formation of programmed DNA double-strand breaks that are repaired through an interaction with a homologous chromosome, culminating in the formation of a joint DNA molecule. Models of recombination and proposed structures of the joint molecule are primarily based on the detection of recombination intermediates in yeasts, in vitro biochemical assays, and genetic data that is difficult to interpret because of overlapping gene functions. Models based on these data are often presented as universal facts for all organisms, when in fact there is little in vivo data available outside of yeasts. In this proposal,I describe experiments that will determine the relative contributions of different recombination models to meiotic crossover and noncrossover formation. I will use the metazoan Drosophila melanogaster and a unique genetic assay that allows the recovery of spontaneous recombination events within a heteroallelic locus that can then be analyzed to determine the arrangement of heteroduplex DNA. I also propose whole genome analysis of recombination events using next generation sequencing. Drosophila has the advantage that it is the only known organism in which a putative double Holliday junction resolvase, MEI-9, that is required for 95% of all crossovers has been identified. By combining the proposed genetic experiments with the already known biochemical properties of MEI-9, I will be able to make strong conclusions about how recombination works in Drosophila. These experiments have the advantage of being carried out in the context of a metazoan germline, and of measuring spontaneous recombination events at higher resolution than other metazoan systems are able to. Results from these experiments will expand knowledge of recombination mechanisms to model systems other than fungi and are directly applicable to both human fertility issues and cancer.