Analysis of replication fork restart and checkpoint regulation after DNA damage

DNA损伤后复制叉重启和检查点调控分析

• 批准号: 7585705
• 负责人: OSCAR M APARICIO
• 金额: $ 32.6万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-02-01 至 2012-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7585705
• 关键词: Adverse effects; Bromodeoxyuridine; CHEK2 gene; Cancer Cell Growth; Cell Cycle Checkpoint; Cell physiology; Cells; Chemical Exposure; Chromosomal Instability; Complex; Condition; Coupling; DNA Damage; DNA Repair; DNA Replication Damage; DNA Sequence; DNA biosynthesis; DNA chemical synthesis; DNA lesion; DNA replication fork; Defect; Detection; Development; Disease regression; Exogenous Factors; Gene Mutation; Genomic Instability; Hereditary Disease; Homeostasis; Lead; Left; Lesion; Malignant Neoplasms; Methods; Mitosis; Modification; Molecular; Molecular Genetics; Mutagenesis; Mutation; Normal Cell; Organism; PCNA gene; Pathway interactions; Phosphorylation; Phosphotransferases; Play; Polymerase; Prevention; Proteins; Public Health; Regulation; Replication-Associated Process; Restart; Role; Saccharomyces cerevisiae; Signal Pathway; Single-Stranded DNA; Stress; Thinking; abstracting; cancer therapy; cell growth; cell injury; chemotherapy; cost; genetic analysis; improved; irradiation; mutant; new technology; polymerization; prevent; repaired; response; ultraviolet irradiation

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.

描述（由申请人提供）： DNA 复制必须以极高的精确度进行，才能保证有机体的正常发育并维持细胞稳态。由于内在和外在因素，如辐射和化学暴露，DNA 不可避免地会发生损伤。 DNA 损伤在 DNA 复制过程中尤其危险，因为它会导致 DNA 复制叉“停滞”，从而导致更严重的 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。复制叉处的 DNA 合成与检查点调节之间的这种直接联系强烈表明，S 内检查点与 DNA 复制协调复制叉重启和 DNA 修复机制，统称为“DNA 损伤耐受”(DDT) 途径。我们建议研究 DDT 在 DNA 损伤反应中停滞的复制叉的重新启动中的参与，以及 S 内检查点对 DDT 的调节。该提案的具体目标是：1）表征复制叉动态响应 DNA 损伤，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常常是由 DNA 突变引起的。突变虽然罕见，但有时会在 DNA 复制过程中发生，DNA 复制是产生生物体整个 DNA 序列的精确副本以产生新细胞的过程。由于癌细胞不受控制的生长依赖于 DNA 复制，因此许多化疗通过破坏 DNA 复制来发挥作用，不幸的是，这对正常细胞生长产生副作用。我们开发了新技术来研究DNA在复制叉处的复制，DNA实际上是在复制叉处复制的。这项研究将研究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。公共健康相关性：项目叙述该提案将研究酿酒酵母 DNA 损伤后复制叉重新启动的调控和分子机制。我们的研究将重点关注 S 内检查点因子 Rad53 在控制复制叉遇到 DNA 损伤的活动以及协调在复制重启和 DNA 修复中发挥作用的 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性疾病的发生。