Control of meiotic double strand break formation

描述（由申请人提供）：在大多数有性生殖的生物体中，配子的形成涉及一个被称为减数分裂重组的受控基因组片段和重组阶段。除了促进遗传多样性外，DNA序列的交换还可以拴住同源染色体，这对于控制染色体分类进入精子或卵子是必不可少的。减数分裂重组是由DNA双链断裂（DSBs）引发的。由于DSB本身难以修复，减数分裂DSB的形成必须严格调节，以防止基因组重排、配子异常和出生缺陷。该项目的总体目标是确定限制减数分裂DSB在适当时间和基因组位置的分子机制，并确定减数分裂DSB不适当形成对DSB修复和基因组稳定性的影响。减数分裂DSB控制将研究在有性生殖酵母酿酒酵母。该项目的初步研究确定了减数分裂DSB活性抑制的两种机制：(i) DSB的形成随着DNA复制的延迟而减弱，（ii） DSB在高度重复的核糖体DNA （rDNA）附近被组成性抑制。这些研究进一步表明，DNA复制和DSB形成之间的耦合是一个专门的检查点机制的结果，这个检查点的一个组成部分已经被确定。拟议的实验将使用分子生物学、遗传学和基因组学方法来定义这个检查点如何调节减数分裂DSB机制，并确定额外的检查点成分。初步研究还发现了rDNA附近抑制dsb所需的保守蛋白，并表明染色体结构在这一过程中起重要作用。本实验将确定rDNA附近的减数分裂染色体结构，并确定该蛋白对DSB机制局部活性的影响。此外，修复中间体的遗传分析和物理分析将用于确定不适当的DSB形成对减数分裂基因组完整性和rDNA重复序列稳定性的影响。

DESCRIPTION (provided by applicant): 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 DNA double strand breaks (DSBs). Because DSBs are inherently difficult to repair, meiotic DSB formation must be tightly regulated to prevent genome rearrangements, aberrant gametes, and birth defects. The overall goal of this project is to define the molecular mechanisms that restrict meiotic DSBs to the appropriate times and genomic locations, and to determine the consequences of inappropriate meiotic DSB formation on DSB repair and genome stability. Meiotic DSB control will be investigated in the sexually reproducing yeast Saccharomyces cerevisiae. Preliminary studies for this project identified two mechanisms of active meiotic DSB suppression: (i) DSB formation is attenuated in response to delayed DNA replication, (ii) DSBs are constitutively suppressed in the vicinity of the highly repetitive ribosomal DNA (rDNA). Those studies furthermore suggested that the coupling between DNA replication and DSB formation is the consequence of a specialized checkpoint mechanism and one component of this checkpoint has been identified. The proposed experiments will use molecular biological, genetic, and genomic approaches to define how this checkpoint regulates the meiotic DSB machinery and to identify additional checkpoint components. Preliminary studies also identified a conserved protein required for the suppression of DSBs in the vicinity of the rDNA and suggested an important role for chromosome structure in this process. The proposed experiments will define the meiotic chromosome structure near the rDNA and determine the effect of this protein on the local activity of the DSB machinery. In addition, genetic assays and physical analysis of repair intermediates will be used to determine the consequences of inappropriate DSB formation on meiotic genome integrity and rDNA repeat stability. PUBLIC HEALTH RELEVANCE: Genome rearrangements and errors in chromosome assortment resulting from inappropriate meiotic recombination are associated with a variety of birth defects, including Down syndrome, Williams syndrome, and Prader-Willi syndrome. By defining the molecular mechanisms that control the initiation of meiotic recombination, this project will provide significant insight into the mechanisms that protect chromosomal integrity during gamete production and will serve as an important framework for the study of birth defects in humans.