Cellular Responses to DNA Replication Stress

细胞对 DNA 复制压力的反应

基本信息

批准号：
8194711
负责人：
Marcus Smolka
金额：
$ 22.88万
依托单位：
CORNELL UNIVERSITY
依托单位国家：
美国
项目类别：
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/8194711
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&apos 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

项目摘要

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. PUBLIC HEALTH RELEVANCE: Our research will contribute to a better understanding of how cancer is initiated. The results could lead to more efficient strategies for cancer management by providing rationale for the development of highly specific drugs.

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，因为复制叉在遇到复制块或损坏的 DNA 模板时很容易停滞或崩溃。为了正确复制基因组，细胞依赖于复制检查点 (RC)，这是一种进化保守的信号通路，持续监控 DNA 复制叉的完整性。根据对临床标本的研究，有人提出 RC 可以构成多种癌症进展的早期屏障，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 中发挥着关键作用。为了应对复制压力，ATR 在受损叉位点快速激活，启动复杂的信号网络，促进叉稳定和修复。尽管很重要，但 ATR 如何调节复制诱导的 DNA 损伤的修复尚不清楚。我们最近在酿酒酵母中的研究揭示了重要的见解，表明 Mec1（酵母 ATR）介导复制因子 Dpb11（人 TopBP1 的直系同源物）与 Slx4（一种协调 DNA 修复因子作用的支架蛋白）的关联。虽然我们的工作将 Dpb11 和 Slx4 置于 RC 介导的叉修复的核心，但这些蛋白质如何协调受损叉处的修复途径的作用仍然是一个悬而未决的问题。此外，由于最近刚刚鉴定出 Slx4 的哺乳动物直系同源物，这种高度保守的支架如何将 RC 信号传导与修复途径连接起来，成为一个基本问题，对理解基因组维护和癌症具有重要意义。为了阐明 RC 信号如何维持分叉完整性的长期目标，在目标 1 中，我们使用酵母遗传学作为强大的工具来定义 RC 信号的 Mec1-Slx4-Dpb11 轴如何控制响应复制块的修复途径。在目标 2 中，我们使用一种新的 Slx4 基因靶向小鼠模型来鉴定哺乳动物 Slx4 在防止复制诱导的基因组不稳定的修复途径中的保守作用和潜在的新作用。我们预计这些研究将确立 Slx4 作为酵母和哺乳动物复制叉修复的关键 RC 效应子。在目标 3 中，我们确定 Dpb11 如何控制 Slx4 和其他修复效应器的使用，以进行病变特异性 DNA 修复，包括修复复制诱导的双链断裂。结果将描述 Slx4 如何在 RC 中发挥作用，并将揭示 Dpb11 在修复途径中以前未被认识到的作用。总而言之，我们期望这里提出的工作将显着增强我们对细胞如何响应复制压力的理解。鉴于 RC 信号传导与癌症的直接关系，以及复制应激作为癌症治疗策略的广泛使用，我们预计我们的工作将对人类健康产生广泛的影响。公共卫生相关性：我们的研究将有助于更好地了解癌症是如何发生的。研究结果可以为开发高度特异性的药物提供理论依据，从而制定更有效的癌症管理策略。