Emerging Mechanisms of Replication-coupled DNA Repair

复制耦合 DNA 修复的新兴机制

基本信息

批准号：
10720698
负责人：
Daniel Semlow
金额：
$ 33.46万
依托单位：
CALIFORNIA INSTITUTE OF TECHNOLOGY
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-08-01 至 2028-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10720698
关键词：
Address Aging Bacteria Biochemical Biochemistry Bone marrow failure Bypass CDC45L gene Cell physiology Cells Cellular biology Chemical Agents Chemical Structure Chemicals Chromatin Chromosomal Rearrangement Cisplatin Coupled DNA DNA Damage DNA Double Strand Break DNA Interstrand Cross-Link Repair DNA Interstrand Crosslinking DNA Repair DNA Repair Disorder DNA biosynthesis DNA glycosylase DNA lesion DNA replication fork DNA-protein crosslink Data Deoxyribonucleases Disease Progression Disparate Ensure Environment Event Exhibits Exposure to Fanconi Anemia pathway Fanconi&apos s Anemia Genetic Diseases Genome Genome Stability Genomic Instability Human Human Genome Induced Mutation Inflammatory Lesion Link MCM2 gene Malignant Neoplasms Maps Mechlorethamine Metabolism Methods Molecular Molecular Biology Mutagens Mutation NEIL3 gene Natural regeneration Nucleotides Outcome Pathway interactions Peptide Hydrolases Physiological Process Proteins Rana Resolution Role S phase Shapes Single-Stranded DNA Site Source Structure Surgical incisions Symptoms Syndrome Therapeutic Therapeutic Intervention Time Toxin Work Xenopus cancer cell cancer predisposition chemotherapy chromosome missegregation clinically relevant crosslink egg experimental study flexibility gene product genome-wide genomic locus gut bacteria helicase homologous recombination insight interest microbiome novel nuclease physical property preference prevent programs recruit repaired replication stress response therapy development tumor progression

项目摘要

Cells are constantly exposed to exogenous and endogenous agents that chemically damage the genome. During DNA replication, this chemical DNA damage can introduce mutations, chromosomal rearrangements, and chromosome mis-segregation events that contribute to progression of cancer and ageing. DNA interstrand cross- links (ICLs) are highly toxic DNA lesions that covalently link the two strands of DNA and block unwinding by the replicative CDC45/MCM2-7/GINS (CMG) helicase. These lesions are generated by cancer chemotherapeutics, endogenous metabolites, and microbiome toxins. Deficits in ICL repair cause the bone marrow failure and cancer predisposition syndrome Fanconi anemia (FA). The products of genes implicated in FA participate in a common ICL repair pathway that is activated when CMG collides with an ICL. Replication fork stalling at the ICL initiates nucleolytic incisions that convert the ICL into a DNA double strand break (DSB). The DSB is itself a potential source of genome instability that must be repaired by homologous recombination. In previous work, we used Xenopus egg extracts to demonstrate that certain ICLs are repaired by an alternative pathway that is also activated upon CMG collision with an ICL. In this pathway, the NEIL3 glycosylase cleaves an N-glycosyl bond in the cross-link, resolving the ICL without DSB formation but generating a labile abasic (AP) site. Our work indicated that the NEIL3 pathway is the preferred response for resolving a subset of ICLs, though the FA pathway can process these lesions when NEIL3 is inactivated. We further showed that the AP site produced by NEIL3 forms a DNA-protein cross-link with the HMCES protein, which stabilizes the AP site and regulates mutagenic DNA synthesis past the AP site. These results indicate that multiple functionally distinct pathways can cooperate to promote efficient replication-coupled repair of DNA damage. In this proposal we will use approaches spanning biochemistry, molecular biology, and cell biology to investigate how repair mechanisms are coordinated at the replication fork during repair of physiologically- and clinically-relevant DNA lesions. In Aim 1, we will determine the mechanisms of repair for an ICL formed by a bacteria toxin implicated in cancer progression, providing new insight as to how the chemical structure of an ICL influences repair. In Aim 2, we will explore the repair of ICLs by the NEIL3/HMCES pathway, including examining how this pathway is activated and how it regulates ICL repair outcomes. In Aim 3, we will examine how HMCES regulates AP site metabolism and contributes to genome stability in cells. These experiments will provide a deeper understanding of how different biochemical repair activities are integrated at stalled replication forks. This work has the potential to inform therapeutic interventions that modulate replication-coupled repair to sensitize cancer cells to chemotherapy or halt progression of diseases caused by DNA repair deficiencies.

细胞不断暴露于对基因组造成化学损伤的外源性和内源性物质。期间 DNA 复制，这种化学 DNA 损伤会引入突变、染色体重排和染色体错误分离事件导致癌症和衰老的进展。 DNA链间交叉链接 (ICL) 是一种剧毒 DNA 损伤，共价连接两条 DNA 链并阻止 DNA 解旋。复制型 CDC45/MCM2-7/GINS (CMG) 解旋酶。这些病变是由癌症化疗产生的，内源代谢物和微生物毒素。 ICL 修复缺陷导致骨髓衰竭和癌症易感综合征范可尼贫血（FA）。与 FA 相关的基因产物参与共同的当 CMG 与 ICL 碰撞时，ICL 修复途径被激活。复制叉在 ICL 启动时停止将 ICL 转化为 DNA 双链断裂 (DSB) 的溶核切口。 DSB本身就是一个潜在的基因组不稳定的根源，必须通过同源重组来修复。在之前的工作中，我们使用了非洲爪蟾卵提取物证明某些 ICL 是通过另一种途径修复的，该途径也 CMG 与 ICL 碰撞时激活。在此途径中，NEIL3 糖基化酶裂解 N-糖基键交联，在不形成 DSB 的情况下解析 ICL，但生成不稳定的脱碱基 (AP) 位点。我们的工作表明 NEIL3 途径是解决 ICL 子集的首选反应，尽管 FA 途径当 NEIL3 失活时，可以处理这些病变。我们进一步证明了NEIL3产生的AP位点与 HMCES 蛋白形成 DNA-蛋白交联，稳定 AP 位点并调节诱变 DNA 合成经过 AP 位点。这些结果表明多个功能不同的途径可以合作促进 DNA 损伤的有效复制耦合修复。在本提案中，我们将使用跨越生物化学、分子生物学和细胞生物学，研究修复机制如何在修复生理和临床相关 DNA 损伤期间的复制叉。在目标 1 中，我们将确定由与癌症进展有关的细菌毒素形成的 ICL 的修复机制，提供了新的深入了解 ICL 的化学结构如何影响修复。在目标 2 中，我们将探索 ICL 的修复通过 NEIL3/HMCES 通路，包括检查该通路如何激活以及它如何调节 ICL 修复结果。在目标 3 中，我们将研究 HMCES 如何调节 AP 位点代谢并有助于细胞中基因组的稳定性。这些实验将更深入地了解不同的生化作用如何修复活动集成在停滞的复制叉上。这项工作有可能为治疗提供信息调节复制偶联修复以使癌细胞对化疗敏感或停止的干预措施 DNA修复缺陷引起的疾病进展。