Checkpoints and Double Strand Breaks in S. Pombe Meiosis

描述（由申请人提供）：基因组不稳定导致减数分裂期间染色体错误分离占人类流产的50%，并导致出生缺陷，如唐氏综合症。正常细胞周期中基因组稳定性的维持与DNA复制叉的稳定性和修复密切相关。这需要由几个高度保守的激酶介导的活跃的检查点信号通路。对模式生物，特别是酵母的研究，为这些激酶在增殖细胞中的作用机制提供了重要的见解。然而，很少有关于“正常”检查点如何在减数分裂S期（meiS）响应复制应激时起作用的研究。相反，大多数减数分裂的研究都集中在DNA复制下游的关键事件上，包括程序性双链断裂的形成、重组和减数分裂I的分裂。因此，在了解减数分裂细胞在meiS阶段如何应对复制应激和DNA损伤方面存在一个关键的空白，这与减数分裂重组程序的激活是分开的。有趣的是，有证据表明，meiS阶段的检查点反应途径被彻底重新编程。它们不是防止DNA损伤，而是促进DNA损伤，从而帮助创造重组的底物，这是染色体分离的必要条件。事实上，S期检查点激酶Cds1 （ScRad53）和复制激酶Hsk1及其亚基Dfp1 （ScCdc7, ScDbf4）通常保持复制叉的完整性并促进DNA修复，而在减数分裂期间积极促进双链断裂的形成。亲本提案解决了细胞如何调节其对复制叉稳定性的敏感性以促进减数分裂分化程序。本补充修订研究了减数分裂中复制叉不稳定的分子后果，通过询问叉崩溃如何影响减数分裂特异性蛋白的募集和分布，包括减数分裂内切酶Rec12Spo11，减数分裂内聚蛋白Rec8，以及将同源物连接在一起的线性元件的成分。我们假设叉折引起的断裂会影响这些蛋白质的分布，从而影响DNA断裂的分布。此次修订将深度测序技术与染色质免疫沉淀（ChIP-seq）相结合，以查询基因组并确定复制胁迫如何影响减数分裂期间这些大分子相互作用。虽然这些方法对我们的实验室来说是新的，但有大量的当地专业知识将帮助我们将其纳入我们的细胞生物学方法。作为这些实验的结果，我们将能够在减数分裂中基因组不稳定的条件下将复制起源，叉崩溃和程序性双链断裂联系起来。这将为影响人类和酵母减数分裂进程的压力提供重要的见解。

DESCRIPTION (provided by applicant): Genome instability resulting in chromosome mis-segregation during meiosis accounts for as many as 50% of human miscarriages and contributes to birth defects such as Down Syndrome. Maintenance of genome stability during the normal cell cycle is closely tied to DNA replication fork stability and repair. This requires active checkpoint signaling pathways mediated by several highly conserved kinases. Studies in model organisms, especially yeasts, has provided critical insights into the mechanisms by which these kinases function in proliferating cells. However, there has been little investigation of how "normal" checkpoints function in meiotic S phase (meiS) in response to replication stress. Instead, most studies of meiosis have focused on key events downstream of DNA replication, including formation of programmed double strand breaks, recombination and the reductional meiosis I division. Thus, there is a critical gap in understanding how meiotic cells respond to replication stress and DNA damage during meiS phase, which is separate from the activation of the meiotic recombination program. Intriguingly, evidence suggests that the checkpoint response pathways in meiS phase are radically reprogrammed. Instead of preventing DNA damage, they actually promote it, and thus help create substrates for recombination, which is necessary for proper chromosome segregation. Indeed, the S phase checkpoint kinase Cds1 (ScRad53) and the replication kinase Hsk1 and its subunit Dfp1 (ScCdc7, ScDbf4) which normally preserve replication fork integrity and promote DNA repair, instead actively promote double strand break formation during meiosis. The parent proposal addresses how the cell adjusts its sensitivity to replication fork stability to facilitate the meiotic differentiation proram. This supplemental revision examines the molecular consequences of replication fork instability in meiosis by asking how fork collapse impacts the recruitment and distribution of meiosis-specific proteins relative to breaks, including the meiotic endonuclease Rec12Spo11, the meiotic cohesin Rec8, and components of the linear elements that link homologues together. We hypothesize that breaks induced by fork collapse will influence the distribution these protein, and thus impact the distribution of DNA breaks. This revision will deep sequencing technologies combined with chromatin immunoprecipitation (ChIP-seq) to query the genome and determine how replication stress impacts these macromolecular interactions during meiosis. While these methods are new to our laboratory, there is substantial local expertise that will help us bring thi on board to complement our cell biology approaches. As a result of these experiments, we will be able to correlate replication origins, fork collapse, and programmed double strand breaks under conditions of genome instability in meiosis. This will provide important insights into the stresses that impact meiotic progression in humans as well as yeast.