Meiotic recombination in Drosophila

描述（由申请人提供）：交叉指导减数分裂染色体的准确分离。减数分裂重组缺陷导致染色体分离异常。在严重的情况下，这会导致不育。当只有一对染色体受到影响时，减数分裂重组的缺失会产生单体和三体。据估计，25-30%的人类受孕由于这些缺陷导致自然终止。尽管广泛的研究，主要是在酿酒酵母中，已经阐明了减数分裂重组途径的许多细节，但我们仍然不知道重组中间体是如何被加工以产生交叉的。很明显，在不同的生物体中，实际产生交叉的过程差异很大，因此开发额外的模型很重要。在黑腹果蝇中，一些同源物在其他途径中具有良好功能的保守蛋白被以新的方式用于产生减数分裂交叉。为了了解果蝇的交叉是如何产生的，我们将结合各种突变体重组产物的遗传和分子分析以及相关蛋白质的生化研究。我们将首先从错配修复突变体中恢复基因内重组事件。我们将使用多个分子标记来确定每个同源物对重组产物的贡献。这将使我们能够区分不同的重组模型。然后，我们将在无法产生交叉的突变体中重复这些实验，包括mei-9和rec。mei-9是果蝇核苷酸切除修复内切酶，需要在减数分裂中产生交叉和进行错配修复。REC， MCM8的果蝇同源基因，也需要产生减数分裂交叉。对这些突变体中重组事件的分析将检验有关这些蛋白质在产生交叉中的功能的假设。我们还将通过纯化昆虫细胞中的复合物和分析模型合成底物上的活性来验证MEI-9和伴侣蛋白切断Holliday连接的假设。这里提出的工作将提供对模型后生动物减数分裂重组机制的更好理解，并将允许对不同生物中的重组机制进行比较。

DESCRIPTION (provided by applicant): Crossovers direct the accurate segregation of meiotic chromosomes. Defects in meiotic recombination lead to aberrant chromosome segregation. In severe cases, this results in sterility. When only one chromosome pair is affected, loss of meiotic recombination can give rise to monosomies and trisomies. It is estimated that 25-30% of human conceptions result in spontaneous termination due to these defects. Although extensive research, primarily in S. cerevisiae, has elucidated many details of the meiotic recombination pathway, we still do not know how recombination intermediates are processed to generate crossovers. It has become evident that the processes that actually generate crossovers vary widely in different organisms, so it is important to develop additional models. In Drosophila melanogaster, several conserved proteins whose homologs have well-characterized functions in other pathways are used in novel ways to generate meiotic crossovers. To understand how crossovers are generated in Drosophila, we will combine genetic and molecular analyses of recombination products from various mutants with biochemical studies of the proteins involved. We will first recover intragenic recombination events from mismatch repair mutants. We will use multiple molecular markers to determine the contribution of each homolog to recombination products. This will allow us to distinguish between different models for recombination. We will then repeat these experiments in mutants that are unable to generate crossovers, including mei-9 and rec. MEI-9 is a Drosophila nucleotide excision repair endonuclease and is required to generate crossovers and conduct mismatch repair in meiosis. REC, the Drosophila ortholog of MCM8, is also required to generate meiotic crossovers. Analysis of recombination events in these mutants will test hypotheses concerning the functions of these proteins in generating crossovers. We will also test the hypothesis that MEI-9 and partner proteins cut Holliday junctions by purifying complexes from insect cells and assaying activities on model synthetic substrates. The work proposed here will provide a greater understanding of meiotic recombination mechanisms in a model metazoan and will allow comparison of recombination mechanisms in different organisms.