Cellular Responses to DNA Replication Stress

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，因为当遇到复制块或受损的DNA模板时，复制叉容易失速或崩溃。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，它不断监测DNA复制分叉的完整性。根据对临床标本的研究，RC被认为是防止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中起关键作用。作为对复制压力的响应，ATR在受损叉的位点迅速激活，启动一个复杂的信号网络，促进叉的稳定和修复。尽管ATR很重要，但人们对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工作揭示了重要的见解，表明Mec1（酵母ATR）介导复制因子Dpb11（人类TopBP1的同源物）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的修复途径的作用仍然是一个悬而未决的问题。此外，由于Slx4的哺乳动物同源基因最近才被发现，这种高度保守的支架如何连接rc信号通路以修复途径成为理解基因组维持和癌症的一个基本问题。为了阐明rc信号传导如何维持叉子完整性的长期目标，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信号传导的Mec1-Slx4-Dpb11轴如何在响应复制阻断时控制修复途径。在Aim 2中，我们使用一个新的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和潜在的新作用。我们预计这些研究将确定Slx4作为酵母和哺乳动物复制叉修复的关键rc效应物。在Aim 3中，我们确定了Dpb11如何控制Slx4和其他修复效应物在损伤特异性DNA修复中的使用，包括修复复制诱导的双链断裂。结果将描述Slx4如何在RC中起作用，并揭示Dpb11在修复途径中以前未被认识到的作用。综上所述，我们期望在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。考虑到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有广泛的影响。

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.