Chromosomal control of meiotic double-strand break formation

项目摘要 在大多数有性生殖的生物中，配子的形成都涉及到一个基因组受控的阶段 分裂和重组称为减数分裂重组。除了促进遗传多样性， DNA序列的交换用于拴系同源染色体，这对于受控的 染色体分配到精子或卵子中。减数分裂重组是由程序性诱导 数以百计的DNA双链断裂（DSB），优先发生在染色体热点。最近 工作已经确定了与减数分裂DSB热点位置相关的染色质修饰。然而，在这方面， 这些修改对实际热点使用情况的预测效果较差，表明存在额外的 决定因素本项目的总体目标是确定减数分裂热点的分子机制 使用.热点使用将在有性繁殖酵母酿酒酵母中进行研究， 在减数分裂重组的许多方面都是成功的模型。在S. 酿酒酵母鉴定了一种保守的染色质调节因子，当突变时， 使用.所提出的实验将确定该调节器在控制DSB水平中的功能， 利用这种突变体改变的DSB景观来确定染色质结构的特征 和DNA拓扑结构保持与热DSB热点特异性相关。此外，初步 实验已经确定了一种特征性的DNA拓扑结构，它似乎可以预测热点的活性。的 拟议的实验将解决这种DNA拓扑结构是否与超螺旋有关，并将研究 几种拓扑敏感酶在影响减数分裂DSB热点活性中的作用。的 拟议的实验还将使用全基因组方法来探测三维构象， 减数分裂染色体，并确定当这些构象改变时对热点使用的影响。 总之，拟议的实验将提供减数分裂染色体结构的高分辨率视图， 定义这种结构在忠实地指导数百个减数分裂DSB形成中的作用， 这是配子形成和繁殖的先决条件。

Project Summary The formation of gametes in most sexually reproducing organisms involves a stage of controlled genome fragmentation and reshuffling known as meiotic recombination. Aside from promoting genetic diversity, the exchange of DNA sequences serves to tether homologous chromosomes, which is essential for controlled chromosome assortment into sperm or eggs. Meiotic recombination is initiated by the programmed induction of hundreds of DNA double-strand breaks (DSBs), which occur preferentially in chromosomal hotspots. Recent work has defined chromatin modifications that correlate with the positions of meiotic DSB hotspots. However, those modifications are poor predictors of actual hotspot usage, indicating the existence of additional determinants. The overall goal of this project is to define the molecular mechanisms governing meiotic hotspot usage. Hotspot usage will be investigated in the sexually reproducing yeast Saccharomyces cerevisiae, which has served as successful model for many aspects of meiotic recombination. Preliminary experiments in S. cerevisiae identified a conserved chromatin regulator that, when mutated, dramatically alters relative hotspot usage. The proposed experiments will determine the function of this regulator in controlling DSB levels, and take advantage of the altered DSB landscape of this mutant to determine the features of chromatin architecture and DNA topology that remain specifically associated with hot DSB hotspots. In addition, preliminary experiments have identified a characterisitic DNA topology that appears to predict hotspot activity. The proposed experiments will address if this DNA topology is related to supercoiling and will investigate the function of several topology-sensitive enzymes in influencing the activity of meiotic DSB hotspots. The proposed experiments will also use genome-wide methods to probe the three-dimensional conformation of meiotic chromosomes and determine the effects on hotspot usage when these conformations are altered. Together, the proposed experiments will provide a high-resolution view of meiotic chromosome structure and define the role of this architecture in faithfully guiding the formation of hundreds of meiotic DSBs as a prerequisite to productive gamete formation and fertility.