Analysis of replication fork restart and checkpoint regulation after DNA damage

描述（由申请人提供）： DNA复制必须以非常精确的方式发生，以允许适当的生物体发育并维持细胞内稳态。不可避免地，DNA损伤是由于内在和外源因素如辐射和化学暴露而发生的。DNA损伤在DNA复制过程中特别危险，因为它会导致DNA复制叉“停滞”，导致更严重的DNA损伤或染色体不稳定。细胞已经进化出复杂的机制来修复DNA损伤，以及细胞周期检查点来抑制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内检查点调节复制叉处的DNA合成，在检查点激活时抑制复制，并在检查点失活时允许复制叉重新启动。叉处的DNA合成与检查点调控之间的这种直接联系强烈表明，S内检查点协调复制叉重启和DNA修复机制，统称为“DNA损伤耐受性”（DDT）途径，与DNA复制。我们建议研究DDT参与重新启动的复制叉，已停止响应DNA损伤，以及调节DDT的内部S检查点。本研究的具体目标是：1）表征DNA损伤后复制叉的动力学，2）研究S内检查点通路在复制叉重启中的作用，3）对复制叉重启中的DDT机制进行分子和遗传分析。摘要：癌症、发育缺陷和其他遗传性疾病经常是由DNA突变引起的。突变虽然罕见，但有时发生在DNA复制过程中，这一过程产生了一个生物体完整DNA序列的精确拷贝，以产生新的细胞。由于癌细胞的不受控制的生长依赖于DNA复制，许多化疗通过破坏DNA复制来起作用，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DNA在复制叉上的复制，DNA实际上是在复制叉上复制的。这项研究将调查调节复制叉的细胞过程，以防止突变或最大限度地减少其潜在的损害。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：项目简介本项目将研究S.啤酒。我们的研究将集中在S内检查点因子Rad53在控制遇到DNA损伤的复制叉的活性和协调在复制重启和DNA修复中起作用的DNA损伤耐受机制中的作用。这些检查点和DNA修复机制对于预防可能导致癌症和其他遗传疾病发展的基因组不稳定性至关重要。

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.