Analysis of replication fork restart and checkpoint regulation after DNA damage

描述(由申请人提供)： DNA复制必须以非常准确的方式进行，才能保证生物的正常发育和维持细胞内环境的稳定。不可避免地，DNA损伤是由于辐射和化学暴露等内源性和外源性因素造成的。DNA损伤在DNA复制过程中尤其危险，因为它会导致DNA复制叉子“熄火”，导致更严重的DNA损伤或染色体不稳定。细胞已经进化出修复DNA损伤的复杂机制，以及细胞周期检查点，以抑制DNA复制和有丝分裂，同时为DNA修复提供机会。我们最近发现，S内部的检查点调节复制分叉的DNA合成，在检查点激活时抑制复制，在检查点停用时允许复制分叉重新启动。分叉处的DNA合成和检查点调控之间的这种直接联系强烈地表明，S内部的检查点协调复制分叉重新启动和DNA修复机制，统称为DNA损伤耐受途径，与DNA复制相协调。我们建议研究滴滴涕在因DNA损伤而停滞的复制叉子重新启动中的作用，以及S内部检查点对滴滴涕的调节。该建议的具体目的是：1)表征复制叉对DNA损伤的响应动力学；2)研究S检查点内通路在复制叉重新启动中的作用；3)对复制叉重新启动中的滴滴涕机制进行分子和遗传学分析。摘要：癌症、发育缺陷和其他遗传性疾病常由DNA突变引起。突变虽然罕见，但有时会发生在DNA复制过程中，DNA复制是指复制生物体的整个DNA序列，从而产生新的细胞的过程。由于癌细胞的不受控制的生长依赖于DNA复制，许多化疗通过扰乱DNA复制来发挥作用，不幸的是，这对正常细胞生长有副作用。我们已经开发了新的技术来研究复制叉处的DNA复制，DNA实际上是在复制的地方复制的。这项研究将研究调节复制分叉的细胞过程，以防止突变或将其潜在损害降至最低。这些研究有可能为癌症的检测和治疗提供改进的方法。公共卫生相关性：项目叙述本提案将调查酿酒酵母DNA损伤后复制分叉重新启动的调节和分子机制。我们的研究将集中在S检查点因子RAD53在控制遇到DNA损伤的复制叉子的活动中的作用，以及在复制重新开始和DNA修复中发挥作用的DNA损伤耐受机制的协调。这些检查点和DNA修复机制对于防止基因组不稳定性至关重要，基因组不稳定性可能导致癌症和其他遗传疾病的发展。

DESCRIPTION (provided by applicant): DNA replication must occur with extraordinary accuracy to permit proper organismal development and to maintain cellular homeostasis. Inevitably, DNA damage occurs due to intrinsic and exogenous factors such as irradiation and chemical exposure. DNA damage is particularly dangerous during the DNA replication process because it can cause DNA replication forks to "stall", leading to even more severe DNA damage or chromosomal instabilities. Cells have evolved complex mechanisms to repair DNA damage, as well as cell cycle checkpoints to inhibit DNA replication and mitosis while providing an opportunity for DNA repair. We have recently found that the intra-S checkpoint regulates DNA synthesis at replication forks, inhibiting replication upon checkpoint activation and permitting replication fork restart upon deactivation of the checkpoint. This direct connection between DNA synthesis at the fork and checkpoint regulation strongly suggests that the intra-S checkpoint coordinates replication fork restart and DNA repair mechanisms, collectively referred to as "DNA-Damage Tolerance" (DDT) pathways, with DNA replication. We propose to study the involvement of DDT in the restart of replication forks that have stalled in response DNA damage, as well as the regulation of DDT by the intra-S checkpoint. The Specific Aims of this proposal are to: 1) Characterize replication fork dynamics in response to DNA damage, 2) Investigate the role of the intra-S checkpoint pathway in replication fork restart, 3) Perform molecular and genetic analysis of DDT mechanisms in replication fork restart. Lay Abstract: Cancers, developmental defects, and other genetic disorders are frequently caused by mutation of DNA. Mutations, though rare, sometimes occur during DNA replication, the process that produces an exact copy of an organism's copies entire DNA sequence to produce new cells. Because the uncontrolled growth of cancer cells depends on DNA replication, many chemotherapies act by disrupting DNA replication, which unfortunately have side-effects for normal cell growth. We have developed new technologies to study DNA replication at replication forks where the DNA is actually copied. This study will investigate the cellular processes that regulate replication forks to prevent mutations or minimize their potential damage. These studies have the potential of providing improved methods for the detection and treatment of cancer. PUBLIC HEALTH RELEVANCE: Project Narrative This proposal will investigate the regulation and molecular mechanisms of replication fork restart after DNA damage in S. cerevisiae. Our studies will focus on the role of intra-S checkpoint factor Rad53 in controlling the activity of replication forks encountering DNA damage and coordination of DNA-damage tolerance mechanisms that function in replication restart and DNA repair. These checkpoint and DNA repair mechanisms are critical to the prevention of genome instabilities that can lead to the development of cancers and other genetic disorders.