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于种系中的基因组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）不稳定性的有力决定因素。减数分裂重组由Spo11蛋白产生的双链断裂（DSB）启动。本研究提出了芽殖酵母S.啤酒。目的是：1）确定确保DNA复制后DSB形成的调控机制。将测试一个工作模型，其中细胞周期调节激酶与复制体的物理关联靶向Mer2蛋白，用于特异性地在已复制的染色质中磷酸化。还将探讨复制和DSB形成之间的全基因组关系。 2)了解决定DSB分布的因素。不同的基因组区域显示出不同的DSB形成倾向，这种“DSB景观”由许多因素的组合和分层作用形成。对这些因素缺乏详细的了解。一种新的方法，在核苷酸分辨率的DSB全基因组映射将被用来确定本地染色质结构和蛋白质参与高阶染色体折叠（凝聚素和染色体轴蛋白）的贡献。此外，不同野生型实验室菌株的DSB图谱将测试DSB分布如何随遗传背景而变化。 3)目的探讨DSB定位与基因转换带的关系。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检测相结合，以围绕一组选定的DSB热点以高空间和定量精度绘制基因转换束。将这些图与高分辨率DSB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性。分散的同源DNA片段之间的分离可导致染色体重排。这种非等位基因重组已在酵母减数分裂中被广泛研究，但通常使用人工重复。在这里，DSB内和附近的天然重复序列（反转录转座子Ty元件）的发生将使用新的全基因组定位方法和物理测定来确定。此外，用于检测和定量非等位Ty元件之间的交换的新方法将用于确定Tys内的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.