Recombination mechanisms of crossover and noncrossover formation in Drosophila

描述（由申请人提供）：为了确保在减数分裂期间染色体的准确分离，使用重组来创建同源染色体之间的交叉。重组是由程序化DNA双链断裂的形成开始的，通过与同源染色体的相互作用修复，最终形成一个联合的DNA分子。重组模型和关节分子的拟议结构主要基于酵母中重组中间体的检测、体外生化分析和由于基因功能重叠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的普遍事实，而事实上，除了酵母之外，几乎没有体内数据可用。在本提案中，我描述了将确定不同重组模式对减数分裂交叉和非交叉形成的相对贡献的实验。我将使用后生动物黑腹果蝇和一种独特的基因分析方法，该方法允许在异等位基因位点内恢复自发重组事件，然后可以分析以确定异双工DNA的排列。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，它是已知的唯一一种已知的生物，在这种生物中，假定的双霍利迪结分解酶MEI-9是95%的杂交所必需的。通过将提出的基因实验与已知的MEI-9的生化特性相结合，我将能够对果蝇的重组如何起作用得出强有力的结论。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展重组机制的知识，以模拟除真菌以外的系统，并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.