Checkpoints and Double Strand Breaks in S. Pombe Meiosis

描述(由申请人提供)：忠实的染色体分离对于生产可行的减数分裂产物是必不可少的。虽然对未受干扰的减数分裂染色体分离的调节是众所周知的，但当细胞在出现意外的DNA损伤的情况下尝试减数分裂时会发生什么，人们却知之甚少。这项建议使用分裂酵母模型系统来研究减数分裂过程中对DNA损伤的反应。裂解酵母是分析细胞周期损伤反应的一个强大系统，它与人类有许多共同的调控基因。以前的研究表明，在细胞增殖过程中阻止细胞周期以应对DNA损伤的检查点系统在减数分裂中不起作用。导致复制叉崩溃的条件似乎与减数分裂进程是一致的。在减数分裂进程和增殖细胞对烷基化损伤的反应之间存在遗传联系，这表明翻译合成聚合酶可能在减数分裂中发挥作用。这些观察表明，减数分裂对DNA损伤的反应在分化过程中基本上是重新编程的。这是一个从ARRA(刺激)资金中资助了一年的当前项目的续展。第一个目的解决的问题是，在减数分裂过程中，损伤检查点激酶CHK1是如何被重新编程的，以便在S阶段对损伤不做出反应。第二个目的是问减数分裂细胞如何适应折叠的复制叉子，这在增殖过程中是致命的。第三个目的是提出跨损伤合成(TLS)聚合酶在减数分裂中的新作用。这是基于两个观察结果：第一，在S时期发挥作用的DDK激酶也调节减数分裂和TLS，第二，该激酶中功能等位基因的分离特异性地破坏了减数分裂和TLS。长期目标是了解MEIS阶段的损伤反应调节是如何被修改的，以使后来的减数分裂事件成为可能。本研究的目的是利用裂殖酵母来剖析减数分裂细胞对复制胁迫和S期损伤的不同反应的分子机制。其基本原理是，了解在减数分裂中促进基因组稳定性的机制将有助于识别影响人类流产和出生缺陷的遗传和环境风险因素。核心假设是，在减数分裂过程中，通常起到保护基因组功能的保守活动被加入，以允许程序性遗传损伤。该项目的预期结果是识别和表征新的分子途径。这些将包括可能在高等真核生物中保守的潜在的新因子。这一积极影响将是在理解分化细胞对DNA损伤和基因组稳定性的反应以及更好地理解减数分裂过程中的风险因素方面取得的根本性进展。 与公共卫生相关：相当大一部分出生缺陷是由减数分裂过程中的染色体缺陷引起的，减数分裂是产生卵子和精子的过程。该项目利用遗传学和细胞生物学在简单酵母中研究减数分裂中的染色体如何受到DNA损伤的保护，DNA损伤可能导致减数分裂缺陷。目标是识别保守的蛋白质，以保护减数分裂细胞免受可能导致出生缺陷的缺陷的影响。

DESCRIPTION (provided by applicant): Faithful chromosome segregation is essential to the production of viable meiotic products. While the regulation of unperturbed meiotic chromosome segregation is well understood, it is less known what happens when cells attempt meiosis in the presence of unexpected DNA damage. This proposal investigates the response to DNA damage in meiosis, using a fission yeast model system. Fission yeast is a powerful system in the analysis of damage response in the cell cycle, sharing many regulatory genes with humans. Previous studies have shown that the checkpoint system that works in proliferating cells to block the cell cycle in response to DNA damage is not functional in meiosis. Conditions that cause replication fork collapse appear to be compatible with meiotic progression. There is a genetic link between meiotic progression and the response of proliferating cells to alkylation damage that suggest translation synthesis polymerases may play a role in meiosis. These observations suggest that the meiotic response to DNA damage is substantially reprogrammed during differentiation. This is a renewal of a current project that has been funded for 1 year from ARRA (stimulus) funding. The first aim addresses the question of how the damage checkpoint kinase, Chk1, is reprogrammed in meiosis so that it does not respond to damage during S phase. The second aim asks how meiotic cells accommodate collapsing replication forks, which would be lethal during proliferation. The third aim proposes a novel role for trans-lesion synthesis (TLS) polymerases in meiosis. This is based on two observations: first, that the DDK kinase which functions during S phase also regulation meiosis and TLS, and second, that a separation of function allele in the kinase specifically disrupts meiotic divisions and TLS. The long term goal is to understand how the regulation of the damage response during meiS phase is modified to enable later meiotic events. The objective is to use fission yeast to dissect the molecular mechanisms that differ in the response to replication stress and S-phase damage in meiotic cells. The rationale is that knowledge of mechanisms that promote genome stability in meiosis will allow identification of genetic and environmental risk factors that impact human miscarriages and birth defects. The central hypothesis is that conserved activities that normally function to protect the genome are co-opted in meiosis to allow programmed genetic damage. The expected outcomes of this project are the identification and characterization of new molecular pathways. These will include potentially novel factors, likely to be conserved in higher eukaryotes. The positive impact will be a fundamental advance in understanding of the response of differentiating cells to DNA damage and genome stability, and a better understanding of risk factors during meiosis. PUBLIC HEALTH RELEVANCE: A substantial fraction of birth defects result from chromosomal defects in meiosis, the process that produces eggs and sperm. This project uses genetics and cell biology in simple yeast to study how chromosomes in meiosis are protected from DNA damage that may contribute to meiotic defects. The goal is to identify conserved proteins that protect meiotic cells from defects that could contribute to birth defects.