Control of meiotic double strand break formation

描述（由申请人提供）：在大多数有性生殖生物中，配子的形成涉及被称为减数分裂重组的受控基因组片段化和重组阶段。除了促进遗传多样性外，DNA序列的交换还可以束缚同源染色体，这对于控制染色体分配到精子或卵子中至关重要。减数分裂重组由DNA双链断裂（DSB）启动。由于DSB固有地难以修复，因此必须严格调控减数分裂DSB的形成以防止基因组重排、异常配子和出生缺陷。该项目的总体目标是确定将减数分裂DSB限制在适当时间和基因组位置的分子机制，并确定不适当的减数分裂DSB形成对DSB修复和基因组稳定性的后果。将在有性繁殖的酿酒酵母中研究减数分裂DSB控制。该项目的初步研究确定了主动减数分裂DSB抑制的两种机制：（i）DSB形成响应于延迟的DNA复制而减弱，（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，并且已经确定了该检查点的一个组成部分。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，并确定其他检查点组件。初步的研究还确定了一个保守的蛋白质所需的抑制DSB在附近的rDNA，并建议在这一过程中的染色体结构的重要作用。拟议的实验将定义rDNA附近的减数分裂染色体结构，并确定这种蛋白质对DSB机制的局部活性的影响。此外，将使用修复中间体的遗传测定和物理分析来确定不适当的DSB形成对减数分裂基因组完整性和rDNA重复稳定性的后果。 公共卫生相关性： 由不适当的减数分裂重组引起的基因组重排和染色体分类错误与多种出生缺陷相关，包括唐氏综合征、威廉姆斯综合征和普拉德-威利综合征。通过定义控制减数分裂重组启动的分子机制，该项目将为配子产生过程中保护染色体完整性的机制提供重要的见解，并将作为人类出生缺陷研究的重要框架。

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.