Function and Regulation of TRAIP at Replisome-Blocking DNA Lesions

项目总结/摘要 每次细胞分裂时，其基因组必须准确而快速地复制。然而，DNA复制可以 被不同类型的DNA损伤所阻断。DNA链间交联（ICL）和DNA-蛋白质交联 （DPC）是复制的特别艰巨的挑战，因为它们阻碍了 复制型CDC 45/MCM 2 -7/GINS（CMG）解旋酶。如果不能修复这些损伤，可能会导致基因组不稳定 和癌症的发展。ICL和DPC主要在S期被感知和修复， 叉在病变处失速。使用爪蟾卵提取物，我们重建了复制偶联的ICL和DPC 修复.在这个系统中，ICL修复是由病变处两个复制叉的会聚启动的。叉 会聚触发CMG的多泛素化，激活两种ICL修复机制之一-NEIL 3 Fanconi贫血（FA）途径。类似地，DPC修复在复制体冲突触发后启动 DPC泛素化，标记交联蛋白被蛋白酶体蛋白水解降解， 蛋白酶SPRTN。因此，泛素信号转导在调节ICL和DPC修复中起关键作用。我们最近 鉴定TRAIP为E3泛素连接酶，负责ICL处停滞的CMG和DPC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 与复制体组成性组装，位于其前缘。这将使TRAIP能够 泛素化复制体遇到的任何蛋白质屏障，包括DPC和邻接的复制体 在ICL。拟议的研究旨在了解TRAIP在这些背景下的机制和调控。 目的1解决了TRAIP如何遍在化复制体障碍，同时避免过早的复制体解体 使用生化重建、单分子成像和结构分析的组合。目的2 研究TRAIP是否也独立于复制体发挥作用，并使用无偏的蛋白质组学和 功能基因组方法来鉴定新的TRAIP调节剂和效应物。我将执行指导 在约翰内斯·沃尔特博士的联合指导下，在哈佛医学院（HMS）进行了这项工作的第一阶段 以及由不同领域的专家科学家组成的咨询委员会。在这次培训期间，我将补充 加上我以前的生化经验，获得了研究DNA损伤和修复的细胞分析技能， 以及学习cryo-EM，质谱和CRISPR筛选技术。这种指导，沿着 HMS充满活力的研究环境和丰富的职业发展资源，将帮助我 我实现了领导一个独立研究项目的目标，在那里我将努力了解细胞的 对DNA损伤的反应和忠实的基因组复制机制。拟议的研究将 加深了我们对复制体如何处理分叉障碍的理解，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 本发明涉及细胞毒性癌症化疗剂的制备方法、细胞毒性癌症化疗剂的制备方法、细胞毒性癌症化疗剂的制备方法和细胞毒性癌症化疗剂的制备方法。

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.