Mechanism of meiotic recombination in yeast

描述(由申请人提供)： 减数分裂期间的同源重组对生殖系基因组的完整性是必不可少的，但也是基因组多样性、进化和(当错误发生时)不稳定性的强大决定因素。减数分裂重组是由Spo11蛋白引起的双链断裂(DSB)启动的。这项建议解决了在萌芽酵母中DSB形成和重组的分子机制。目的是：1)确定确保DNA复制后形成DSB的调控机制。将测试一个工作模型，在该模型中，细胞周期调节激酶与复制体的物理联系以Mer2蛋白为目标，特别是在已复制的染色质中进行磷酸化。复制和DSB形成之间的全基因组关系也将被探索。2)了解决定DSB分布的因素。不同的基因组区域表现出不同的DSB形成倾向，这种“DSB景观”是由许多因素的组合和分级作用形成的。对这些因素缺乏详细的了解。一种在核苷酸分辨率下绘制DSB全基因组图谱的新方法将用于确定局部染色质结构和参与高阶染色体折叠的蛋白质(粘附素和染色体轴蛋白质)的贡献。此外，不同野生型实验室菌株的DSB图谱将测试DSB分布如何随着遗传背景的变化而变化。3)确定DSB定位与基因转换区的关系。经典的四分体分析将与最先进的基因分型方法和新的物理分析相结合，以高空间和定量的精度绘制围绕一组选定的DSB热点的基因转换区。将这些地图与高分辨率DSB地图进行比较，将测试不同重组模型的预测。4)确定重复序列中的DSB如何导致基因组不稳定。分散的同源DNA片段之间的重组可导致染色体重排。这种非等位基因重组在酵母减数分裂中得到了广泛的研究，但通常是通过人工重复进行的。在这里，将使用新的全基因组作图方法和物理分析来确定在自然重复序列(反转录转座子Ty元件)内和附近出现DSB的情况。此外，还将使用一种新的方法来检测和量化非等位基因Ty元素之间的交叉，以确定TY中的DSB对总染色体重排的贡献程度。

DESCRIPTION (provided by applicant): Homologous recombination during meiosis is essential for genome integrity in the germ line, but is also a powerful determinant of genome diversity, evolution, and (when mistakes occur) instability. Meiotic recombination is initiated by double-strand breaks (DSBs) made by the Spo11 protein. This proposal addresses molecular mechanisms underlying DSB formation and recombination in the budding yeast, S. cerevisiae. Aims are: 1) To define regulatory mechanisms that ensure that DSBs form after DNA replication. A working model will be tested, in which physical association of a cell cycle regulatory kinase with the replisome targets Mer2 protein for phosphorylation specifically in chromatin that has been replicated. The genome-wide relationship between replication and DSB formation will also be explored. 2) To understand factors that determine DSB distributions. Different genomic regions show different propensity for DSB formation, with this "DSB landscape" shaped by combinatorial and hierarchical action of many factors. Detailed understanding of these factors is lacking. A novel method for genome-wide mapping of DSBs at nucleotide resolution will be used to determine the contribution of local chromatin structure and of proteins involved in higher-order chromosome folding (cohesins and chromosome axis proteins). Additionally, DSB maps in divergent wild-type laboratory strains will test how DSB distributions vary with genetic background. 3) To determine the relationship between DSB location and gene conversion tracts. Classical tetrad analysis will be combined with state-of-the-art genotyping methods and novel physical assays to map gene conversion tracts with high spatial and quantitative precision around a set of selected DSB hotspots. Comparing these maps to high resolution DSB maps will test predictions of different recombination models. 4) To determine how DSBs in repetitive sequences contribute to genome instability. Recombination between dispersed homologous DNA segments can lead to chromosome rearrangements. Such non-allelic recombination has been extensively studied in yeast meiosis, but usually with artificial repeats. Here, the occurrence of DSBs within and near natural repeats (retrotransposon Ty elements) will be determined using novel genome-wide mapping methods and physical assays. In addition, a new method for detecting and quantifying crossing over between non- allelic Ty elements will be used to determine the extent to which DSBs within Tys contribute to gross chromosomal rearrangements.