Cellular Responses to DNA Replication Stress

细胞对 DNA 复制压力的反应

• 批准号: 8535170
• 负责人: Marcus Smolka
• 金额: $ 22万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-01 至 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8535170
• 关键词: BRCT Domain; Breast; Cell Survival; Cells; Clinical; Collaborations; Colon; DNA Damage; DNA Repair; DNA biosynthesis; DNA lesion; DNA replication fork; Development; Embryo; Fibroblasts; Gene Targeting; Genetic Recombination; Genome; Genomic Instability; Goals; Health; Heart; Human; Investigation; Lead; Lesion; Link; Maintenance; Malignant Neoplasms; Mammals; Maps; Mediating; Mediator of activation protein; Modeling; Molecular; Monitor; Mus; Orthologous Gene; Pathway interactions; Pharmaceutical Preparations; Phosphatidylinositols; Phosphotransferases; Play; Proteins; Proteomics; Recruitment Activity; Research; Role; Saccharomyces cerevisiae; Saccharomycetales; Scaffolding Protein; Seckel syndrome; Seckel' s Syndrome; Signal Pathway; Signal Transduction; Site; Specificity; Specimen; Stress; Testing; Work; Workplace; Yeasts; base; biological adaptation to stress; cancer therapy; insight; lung Carcinoma; mouse model; mutant; nervous system disorder; novel; prevent; repaired; response; scaffold; tool; tumorigenesis; yeast genetics

DESCRIPTION (provided by applicant): Genomic instability is a threat to cell survival and a major factor that drives tumorigenesis. During DNA replication, cells are particularly vulnerable to accumulate genomic instability as replication forks are prone to stall or collapse when encountering replication blocks or damaged DNA templates. To properly replicate the genome, cells rely on the replication checkpoint (RC), an evolutionary conserved signaling pathway that is constantly monitoring the integrity of DNA replication forks. Based on studies with clinical specimens, the RC has been proposed to constitute an early barrier against the progression of a number of cancers, including carcinomas of the lung, breast and colon. The phosphatidyl-inositol-3-kinase-like kinase ATR plays pivotal roles in the RC. In response to replication stress, ATR is rapidly activated at sites of damaged forks to initiate an elaborate signaling network that promotes fork stabilization and repair. Despite the importance, how ATR regulates the repair of replication-induced DNA lesions is not well understood. Important insights were revealed by our recent work in S. cerevisiae showing that Mec1 (yeast ATR) mediates the association of the replication factor Dpb11 (ortholog of human TopBP1) with Slx4, a scaffold protein that coordinates the action of DNA repair factors. While our work places Dpb11 and Slx4 at the heart of RC-mediated fork repair, how these proteins coordinate the action of repair pathways at damaged forks remains a wide open question. Furthermore, as the mammalian ortholog of Slx4 was just recently identified, how this highly conserved scaffold links RC-signaling to repair pathways emerges as a fundamental problem with implications for understanding genome maintenance and cancer. With the long-term goal of elucidating how RC-signaling maintains fork integrity, in Aim 1 we use yeast genetics as a powerful tool to define how the Mec1-Slx4-Dpb11 axis of RC-signaling controls repair pathways in response to replication blocks. In Aim 2, we use a new Slx4 gene-targeted mouse model to identify both conserved and potentially novel roles for mammalian Slx4 in repair pathways that prevent replication-induced genomic instability. We anticipate that these studies will establish Slx4 as a key RC-effector for replication fork repair in yeast and mammals. In Aim 3 we determine how Dpb11 controls the use of Slx4 and other repair effectors for lesion-specific DNA repair, including the repair of replication-induced double stranded breaks. The results will delineate how Slx4 functions in the RC and will unmask previously unappreciated roles for Dpb11 in repair pathways. Taken together, we expect that the work being proposed here will significantly enhance our understanding of how cells respond to replication stress. Given the direct relationship of RC-signaling with cancer, and the wide-spread use of replication stress as a strategy for cancer therapy, we expect our work to have broad implications for human health.

描述（由申请人提供）：基因组不稳定性是对细胞存活的威胁，也是驱动肿瘤发生的主要因素。在DNA复制过程中，细胞特别容易积累基因组不稳定性，因为当遇到复制阻断或受损的DNA模板时，复制叉容易停滞或崩溃。为了正确地复制基因组，细胞依赖于复制检查点（RC），这是一种进化上保守的信号通路，它不断地监测DNA复制叉的完整性。基于对临床标本的研究，RC已被提议构成对许多癌症的进展的早期屏障，包括肺癌、乳腺癌和结肠癌。磷脂酰肌醇-3-激酶样激酶ATR在RC中起关键作用。为了应对复制应激，ATR在受损叉的位点被迅速激活，以启动一个精心设计的信号网络，促进叉的稳定和修复。尽管重要性，ATR如何调节复制诱导的DNA损伤的修复还没有很好地理解。我们最近在S.显示Mec 1（酵母ATR）介导复制因子Dpb 11（人TopBP 1的直系同源物）与Slx 4（一种协调DNA修复因子作用的支架蛋白）的缔合。虽然我们的工作将Dpb 11和Slx 4置于RC介导的分叉修复的核心，但这些蛋白质如何协调受损分叉处修复途径的作用仍然是一个悬而未决的问题。此外，由于Slx 4的哺乳动物直系同源物最近才被鉴定出来，这种高度保守的支架如何将RC信号传导与修复途径联系起来，成为理解基因组维持和癌症的一个基本问题。为了阐明RC信号如何保持叉完整性的长期目标，在Aim 1中，我们使用酵母遗传学作为一个强大的工具来定义RC信号的Mec 1-Slx 4-Dpb 11轴如何控制修复途径以响应复制阻断。在目标2中，我们使用一种新的Slx 4基因靶向小鼠模型来确定哺乳动物Slx 4在修复途径中的保守和潜在的新作用，这些修复途径可以防止复制诱导的基因组不稳定性。我们预期这些研究将确立Slx 4作为酵母和哺乳动物中复制叉修复的关键RC效应子。在目标3中，我们确定Dpb 11如何控制Slx 4和其他修复效应子用于损伤特异性DNA修复，包括修复复制诱导的双链断裂。结果将描述Slx 4如何在RC中发挥作用，并揭示Dpb 11在修复途径中以前未被认识到的作用。综上所述，我们希望这里提出的工作将大大提高我们对细胞如何应对复制压力的理解。鉴于RC信号与癌症的直接关系，以及复制应激作为癌症治疗策略的广泛使用，我们希望我们的工作对人类健康具有广泛的影响。