Genetic Analysis of MSM Protein Function in S. Pombe

描述（由申请人提供）：准确的DNA复制和基因组完整性的维持对细胞存活至关重要。DNA复制压力与癌症的发展有着特殊的联系。恶性肿瘤伴随着基因组完整性的逐渐丧失，这可能导致或加剧疾病。编码保守的MCM解旋酶的基因失调发生在癌细胞中，MCM突变也与小鼠的癌症发展有关。这些研究表明，分析MCM复合物促进基因组稳定性的机制与人类健康和疾病直接相关。本研究采用一个易于处理的模型系统，即裂变酵母S. pombe，来研究MCM蛋白对染色体完整性的贡献。众所周知，mcm突变体会导致复制分叉崩溃。这表明MCM解旋酶在细胞周期中对复制叉的稳定性和染色体动力学起着至关重要的作用。该项目使用遗传学、分子生物学和细胞生物学方法，包括一个新的单细胞分析系统，以提供稳定和恢复动态的活细胞成像。第一个目标是分析正常、停滞和崩溃分叉的复制分叉组件。它比较了检查点突变中的叉崩溃与MCM解旋酶突变引起的叉崩溃。本目的目的是使用分子方法检查停滞或折叠叉的结构，并通过使用新的染色质纤维技术成像叉蛋白来补充这一点。在目标2中，活细胞分析被用来通过评估损伤反应和修复蛋白对受损叉的招募来检查叉恢复的动力学。它将这与恢复细胞周期的事件联系起来，并检查如果这些反应不正常，细胞分裂是如何中断的。活细胞分析是补充分子方法来检查修复和恢复蛋白在个别复制叉。Aim 3采用分子方法研究MCM复合物，特别是Mcm4是复制检查点激酶Cds1的底物的证据。使用Aim 1和2的方法，分析破坏检查点反应的Mcm4突变形式对蛋白质募集和细胞周期动力学的影响，并检查它们是否影响与复制复合体、检查点蛋白或修复蛋白的关联。目的4检查基因组的特定区域是否增加了对MCM缺陷的敏感性。该目的使用基因组方法来确定mcm突变体中的复制缺陷是否会导致基因组中随机分布的损伤。作为基因组学研究的补充，本研究得出结论，假设核糖体DNA可能对mcm诱导的叉型塌陷特别敏感，并研究是否可以作为mcm依赖性基因组完整性的模型区域。总之，这些目标提供了复制蛋白网络如何相互作用以维持细胞中基因组稳定性的整体图景。公共卫生相关性：当细胞遭受影响染色体稳定性的DNA复制缺陷时，癌症就会发生。一个由保守蛋白组成的网络通常起保护细胞免受DNA损伤和复制缺陷的作用，而这些蛋白的突变与人类的癌症直接相关。这个项目在一个简单的酵母中使用遗传学和细胞生物学来研究这些保守的蛋白质通常是如何保护基因组的，并研究当它们被破坏时的分子后果。

DESCRIPTION (provided by applicant): Accurate DNA replication and maintenance of genome integrity are essential for cell survival. DNA replication stress has been specifically associated with development of cancer. Malignancy is accompanied by progressive loss of genome integrity which can cause or exacerbate the disease. Dysregulation of the genes encoding the conserved MCM helicase occurs in cancer cells, and mcm mutations are also linked to cancer development in mice. These studies indicate that analysis of the mechanism by which the MCM complex contributes to genome stability have direct relevance to human health and disease. This proposal uses a tractable model system, the fission yeast S. pombe, to investigate the contributions of MCM proteins to chromosome integrity. It is known that mcm mutants cause replication fork collapse. This suggests that the MCM helicase is a crucial player in replication fork stability and chromosome dynamics during the cell cycle. The project uses genetics, molecular biology, and cell biology methods, including a new system for single-cell analysis to provide live cell imaging of the dynamics of stabilization and recovery. The first aim analyzes replication fork components at normal, stalled and collapsed forks. It compares fork collapse in checkpoint mutants to that induced by mutation of the MCM helicase. This aim examines the structure of the stalled or collapsed forks using molecular methods, and complements this by imaging fork proteins using a novel chromatin fiber technique. In Aim 2, live cell analysis is used to examine the dynamics of fork recovery by assessing the recruitment of damage response and repair proteins to the damaged fork. It correlates this to events of the recovering cell cycle, and examines how cell division is disrupted if these responses are not normal. The live cell analysis is complemented by molecular approaches to examine repair and recovery proteins at individual replication forks. Aim 3 takes a molecular approach to investigate evidence that the MCM complex, and particularly Mcm4, is a substrate of the replication checkpoint kinase Cds1. It analyzes mutant forms of Mcm4 that disrupt checkpoint response for their effects on protein recruitment and cell cycle dynamics, using the methods of Aim 1 and 2, and examines whether they affect association with the replication complex, checkpoint proteins, or repair proteins. Aim 4 examines whether particular regions of the genome have increased sensitivity to MCM defects. This aim uses genomic approaches to determine whether replication defects in mcm mutants cause stochastically distributed damage in the genome. Complementing the genomics studies, this aim concludes with the hypothesis that the ribosomal DNA may be particularly sensitive to mcm induced fork collapse, and investigates whether can be used as a model region for MCM-dependent genome integrity. Together, these aims provide a holistic picture of how a network of replication proteins interacts to maintain genome stability in the cell. PUBLIC HEALTH RELEVANCE: Cancer results when cells suffer defects in DNA replication that affect chromosome stability. A network of conserved proteins normally functions to protect cells from DNA damage and replication defects, and mutations in these proteins are directly associated with cancer in humans. This project uses genetics and cell biology in a simple yeast to study how these conserved proteins normally work to protect the genome, and to study the molecular consequences when they are disrupted.