Function and Regulation of TRAIP at Replisome-Blocking DNA Lesions

项目摘要/摘要 细胞每次分裂时，其基因组都必须准确而快速地复制。然而，DNA复制可以 被不同类型的DNA损伤所阻挡。DNA链间交联物和DNA-蛋白质交联物 (DPC)对复制来说是特别艰巨的挑战，因为它们阻碍了 复制CDC45/MCM2-7/GINS(CMG)解旋酶。不能修复这些损伤可能会导致基因组不稳定 和癌症的发展。ICL和DPC主要在S阶段复制时被感知和修复 叉子在损伤处停滞不前。利用非洲爪哇卵提取物，我们重组了复制偶联的ICL和DPC 修理。在这个系统中，ICL修复是由两个复制叉子在病变处的会聚启动的。叉 融合触发CMG的多泛素化，激活两种ICL修复机制之一-NEIL3 Fanconi贫血(FA)途径。同样，DPC修复是在复制体冲突触发后启动的 DPC泛素化，标记交联蛋白被蛋白酶体和 蛋白酶SPRTN。因此，泛素信号在调节ICL和DPC修复中起着关键作用。我们最近做了 确定TRAIP是E3泛素连接酶，负责ICL和DPC停滞的CMG的泛素化。 TRAIP如何在这些不同的情况下采取行动仍不清楚。我们发现TRAIP是 复制体与复制体组装在一起，位于其前缘。这将允许TRAIP 泛素化复制体遇到的任何蛋白质屏障，包括DPC和毗邻复制体 在ICL。拟议的研究试图了解TRAIP在这些背景下的机制和调节。 目标1阐述了TRAIP如何在避免复制体早拆的同时泛素化复制体障碍 采用生化重建、单分子成像和结构分析相结合的方法。目标2 研究TRAIP是否也独立于复制体发挥作用，并使用无偏见的蛋白质组和 识别新的TRAIP调节因子和效应因子的功能基因组方法。我会表演被指导的 在约翰尼斯·沃尔特博士的共同指导下，哈佛医学院(HMS)这一阶段的工作 以及一个由不同领域的专家科学家组成的咨询委员会。在此培训期间，我将添加 根据我之前的生化经验，在细胞分析方面获得了调查DNA损伤和修复的技能， 以及学习冷冻-EM、质谱学和CRISPR筛查技术。这种良师益友， 在HMS充满活力的研究环境和丰富的职业发展资源，将有助于我 实现我的目标，领导一个独立的研究项目，在那里我将努力了解细胞的 对DNA损伤的反应和忠实基因组复制的机制。拟议的研究将 加深我们对复制体如何处理阻碍分叉的障碍的理解，并将揭示癌细胞如何 对化疗引起的损伤有反应。这些见解可能会导致治疗DNA修复的新方法 这涉及到缺乏疾病和加强或重新激活细胞毒性癌症化疗药物的新策略。

Project Summary/Abstract Each time a cell divides, its genome must be accurately and rapidly duplicated. However, DNA replication can be blocked by diverse types of DNA damage. DNA interstrand crosslinks (ICLs) and DNA-protein crosslinks (DPCs) are especially formidable challenges to replication because they impede the progression of the replicative CDC45/MCM2-7/GINS (CMG) helicase. Failure to repair these lesions can lead to genomic instability and cancer development. ICLs and DPCs are primarily sensed and repaired during S phase when replication forks stall at the lesion. Using Xenopus egg extracts, we have reconstituted replication-coupled ICL and DPC repair. In this system, ICL repair is initiated by the convergence of two replication forks at the lesion. Fork convergence triggers polyubiquitylation of the CMGs, activating one of two ICL repair mechanisms – the NEIL3 pathway or the Fanconi anemia (FA) pathway. Similarly, DPC repair is initiated after replisome collision triggers DPC ubiquitylation, marking the crosslinked protein for proteolytic degradation by the proteasome and the protease SPRTN. Thus, ubiquitin signaling plays a key role in regulating ICL and DPC repair. We have recently identified TRAIP as the E3 ubiquitin ligase responsible for ubiquitylation of CMGs stalled at ICLs and of DPCs. How TRAIP is regulated to act in these diverse situations remains unclear. We have found that TRAIP is constitutively assembled with the replisome, positioned at its leading edge. This would allow TRAIP to ubiquitylate any proteinaceous barrier encountered by the replisome, including DPCs and abutting replisomes at an ICL. The proposed studies seek to understand the mechanisms and regulation of TRAIP in these contexts. Aim 1 addresses how TRAIP ubiquitylates replisome barriers while avoiding premature replisome disassembly using a combination of biochemical reconstitution, single-molecule imaging, and structural analysis. Aim 2 investigates whether also TRAIP functions independently of the replisome and uses unbiased proteomic and functional genomic approaches to identify novel TRAIP regulators and effectors. I will perform the mentored phase of this work at Harvard Medical School (HMS) under the combined mentorship of Dr. Johannes Walter and an assembled advisory committee of expert scientists in diverse fields. During this training period, I will add to my previous biochemical experience, gaining skills in cellular assays to investigate DNA damage and repair, as well as learning cryo-EM, mass spectrometry, and CRISPR screening techniques. This mentorship, along with the dynamic research environment and abundant career development resources at HMS, will help me realize my goal of leading an independent research program, where I will work toward understanding the cell’s response to DNA damage and the mechanisms of faithful genome duplication. The proposed research will deepen our understanding of how replisomes handle fork-stalling barriers and will shed light on how cancer cells respond to chemotherapy-induced lesions. These insights may lead to new ways of treating DNA repair deficiency disorders and to new strategies to potentiate or reactivate cytotoxic cancer chemotherapeutics.