Cellular Responses to DNA Replication Stress

描述(由申请人提供)：基因组不稳定是对细胞生存的威胁，也是推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的不稳定性，因为当遇到复制障碍或DNA模板损坏时，复制叉容易停滞或崩溃。为了正确复制基因组，细胞依赖于复制检查点(RC)，这是一条进化保守的信号通路，不断监测DNA复制分叉的完整性。根据对临床标本的研究，RC被认为是阻止一些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中起着关键作用。作为对复制压力的响应，ATR在叉子受损的位置被迅速激活，以启动一个复杂的信号网络，促进叉子的稳定和修复。尽管ATR很重要，但它如何调控复制诱导的DNA损伤的修复还不是很清楚。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TopBP1的同源基因)与SLx4的关联，Slx4是一种协调DNA修复因子作用的支架蛋白。虽然我们的工作将Dpb11和SLx4置于RC介导的叉子修复的核心，但这些蛋白质如何协调受损叉子上的修复路径的行动仍然是一个广泛的悬而未决的问题。此外，由于SLX4的哺乳动物同源基因最近才被发现，这个高度保守的支架如何将RC信号连接到修复途径，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信号如何维持分叉的完整性，在目标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Mec1-slx4-Dpb11轴如何控制修复通路以响应复制块。在目标2中，我们使用一种新的SLX4基因靶向小鼠模型来确定哺乳动物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角色。我们预计这些研究将使SLX4成为酵母和哺乳动物复制分叉修复的关键RC效应因子。在目标3中，我们确定了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，包括复制诱导的双链断裂的修复。结果将描绘SLX4如何在RC中发挥作用，并将揭开Dpb11在修复途径中以前未被认识的角色。综上所述，我们预计这里提出的工作将显著增强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，我们预计我们的工作将对人类健康产生广泛的影响。

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.