Emerging Mechanisms of Replication-coupled DNA Repair

细胞不断地暴露在化学损害基因组的外源性和内源性因素中。在.期间 DNA复制，这种化学DNA损伤会导致突变、染色体重排和 导致癌症进展和衰老的染色体错误分离事件。DNA链间交叉- 连接(ICL)是一种剧毒的DNA损伤，它以共价方式连接DNA的两条链，并通过 复制CDC45/MCM2-7/GINS(CMG)解旋酶。这些损伤是由癌症化疗药物产生的， 内源性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有关的基因产物参与了一个共同的 当CMG与ICL发生碰撞时激活的ICL修复途径。ICL启动复制分叉停滞 将ICL转化为DNA双链断裂(DSB)的核溶解切口术。DSB本身就是一个潜在的 基因组不稳定的来源，必须通过同源重组来修复。在之前的工作中，我们使用了 非洲爪哇卵提取物证明某些ICL是通过另一种途径修复的，这也是 在CMG与ICL发生碰撞时激活。在此途径中，NEIL3糖基酶裂解N-糖基键。 这种交联会在没有形成DSB的情况下分解ICL，但会产生不稳定的碱性(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被灭活时，可以处理这些损伤。我们进一步证明了由NEIL3产生的AP位点 与HMCES蛋白形成DNA-蛋白质交联物，稳定AP位点，调节突变 DNA合成超过了AP的位置。这些结果表明，多条功能不同的通路可以协同工作 以促进DNA损伤的高效复制偶联修复。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，以研究修复机制是如何在 在生理和临床相关的DNA损伤修复过程中的复制分叉。在目标1中，我们将确定 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 洞察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途径，包括研究该途径是如何被激活的，以及它是如何调节ICL的 修复结果。在目标3中，我们将研究HMCES如何调节AP部位的新陈代谢并有助于 细胞内基因组的稳定性。这些实验将使我们更深入地了解不同的生物化学 修复活动在停滞的复制分叉上集成。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的进展。

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.