Emerging Mechanisms of Replication-coupled DNA Repair

细胞不断暴露于化学损伤基因组的外源性和内源性试剂。期间 DNA复制，这种化学DNA损伤可以引入突变，染色体重排， 染色体错误分离事件，导致癌症和衰老的进展。DNA链间交叉 连接（ICL）是高度毒性的DNA损伤，其共价连接DNA的两条链并阻断DNA的解旋。 复制型CDC 45/MCM 2 -7/GINS（CMG）解旋酶。这些病变是由癌症化疗药物产生的， 内源性代谢物和微生物组毒素。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有关的基因产物参与了一个共同的 当CMG与ICL碰撞时激活的ICL修复途径。复制分叉在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 必须通过同源重组修复的基因组不稳定性的来源。在以前的工作中，我们使用 非洲爪蟾卵提取物，以证明某些ICL是通过替代途径修复的， 在CMG与ICL碰撞时激活。在该途径中，NEIL 3糖基化酶切割N-糖基键， 交联，在没有DSB形成的情况下分解ICL，但产生不稳定的脱碱基（AP）位点。我们的工作 表明NEIL 3途径是解决ICL子集的首选反应，尽管FA途径 当NEIL 3失活时可以处理这些病变。我们进一步表明，NEIL 3产生的AP位点 与HMCES蛋白形成DNA-蛋白质交联，其稳定AP位点并调节致突变性 DNA合成通过AP位点。这些结果表明，多个功能不同的途径可以合作， 以促进DNA损伤的有效复制偶联修复。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，研究修复机制是如何协调的， 复制叉在生理和临床相关的DNA损伤修复过程中。在目标1中，我们将确定 修复由与癌症进展有关的细菌毒素形成的ICL的机制， 关于ICL的化学结构如何影响修复的见解。在目标2中，我们将探索ICLs的修复 通过NEIL 3/HMCES通路，包括检查该通路如何激活以及如何调节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 细胞中的基因组稳定性。这些实验将提供一个更深入的了解如何不同的生化 修复活动被集成在停止的复制分叉处。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 由DNA修复缺陷引起的疾病进展。

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.