Control of meiotic double strand break formation

描述(申请人提供)：在大多数有性生殖生物体中，配子的形成涉及一个被称为减数分裂重组的受控基因组碎裂和重新洗牌阶段。除了促进遗传多样性，DNA序列的交换还用于系住同源染色体，这对于受控的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(DSB)启动的。由于DSB天生难以修复，减数分裂DSB的形成必须受到严格的调控，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将减数分裂DSB限制在适当的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影响。减数分裂DSB的控制将在有性生殖酵母酿酒酵母中进行研究。本项目的初步研究确定了两种抑制减数分裂活跃的DSB的机制：(I)DSB的形成因DNA复制延迟而减弱，(Ii)DSB在高度重复的核糖体DNA(RDNA)附近受到结构性抑制。这些研究进一步表明，DNA复制和DSB形成之间的耦合是一种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该检查点如何调节减数分裂DSB机制，并确定其他检查点组件。初步研究还发现了在rDNA附近抑制DSB所需的一种保守蛋白质，并表明染色体结构在这一过程中起着重要作用。拟议的实验将定义rDNA附近的减数分裂染色体结构，并确定该蛋白质对DSB机制局部活性的影响。此外，遗传分析和修复中间产物的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重复稳定性的影响。 公共卫生相关性： 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有关，包括唐氏综合征、威廉姆斯综合征和普拉德-威利综合征。通过确定控制减数分裂重组启动的分子机制，该项目将为在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，并将作为研究人类出生缺陷的重要框架。

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.