The Regulation of DNA Repair by Anchored and Unanchored Ubiquitin Chains

项目摘要/摘要 背景：有大量证据证明泛素结合和去结合在DNA修复机制中的重要性，以及一些泛素连接酶和一些脱泛素酶的身份。然而，泛素化的底物在很大程度上仍然存在争议，涉及的泛素链的异质性还没有明确定义。最近，未锚定的泛素链已成为天然免疫和激酶调节的功能调节者，但它们尚未在DNA修复机制中得到解决。了解DNA修复机制的“泛素结构”的全貌是很重要的，探索非锚定泛素链的功能重要性提供了一个迄今被忽视的机会。 目的：这项建议的目的是进一步描述DNA修复机制的“泛素结构”，以便能够利用它们来制定更成功的治疗策略。一方面，将评估自由的、未锚定的泛素链的可能性，以及持续的泛素链形成对DNA修复机制的影响，这两者都将强调去泛素酶(DUB)作为DNA修复途径关键调节因子的重要性。 具体目标：(1)确定DNA损伤反应中非锚定泛素链的存在和丰度，(2)确定持续的泛素链形成对DNA损伤反应的影响，(3)确定DNA损伤灶内替代泛素连接的重要相互作用蛋白。 研究设计：首先，从DNA损伤剂处理的细胞中提纯多泛素结合物，并评估其游离泛素链的含量。然后，我最近开发的一种具有结合能力但去结合缺陷的泛素突变体的效果将被测试，以在没有DNA损伤的情况下启动DNA修复反应机制。此外，将在DNA损伤条件下测试突变体在制造特定连接的泛素链方面的效果，以确定持续的泛素链形成是否会使细胞对DNA损伤敏感或抑制反应。最后，通过亲和纯化从DNA损伤诱导的细胞中纯化泛素链结合蛋白，通过质谱学鉴定参与DNA损伤的替代泛素链的新的相互作用蛋白。

Project Summary/Abstract Background: There is a considerable amount of evidence documenting the importance of ubiquitin conjugation and deconjugation in DNA repair mechanisms, as well as the identity of some the ubiquitin ligases and some of the deubiquitinating enzymes involved. However, the substrates of ubiquitination are still largely controversial and the heterogeneity of the ubiquitin chains involved has not been clearly defined. Recently, unanchored ubiquitin chains have emerged as functional regulators of innate immunity and kinase regulation, however they have not yet been addressed in DNA repair mechanisms. It is important to understand the complete picture of the "ubiquitin architecture" of DNA repair mechanisms and exploring the functional importance of unanchored ubiquitin chains presents a hitherto overlooked opportunity. Objective: The goal of this proposal is to further characterize the "ubiquitin architecture" of DNA repair mechanisms in order to be able to exploit them for more successful therapeutic strategies. On the one hand, the possibility of free, unanchored ubiquitin chains will be assessed, as well as the effect of sustained ubiquitin chain formation on DNA repair mechanisms, both of which will underscore the importance of deubiquitinating enzymes (DUBs) as crucial regulators of DNA repair pathways. Specific Aims: (1) Determine the existence and abundance of unanchored ubiquitin chains during DNA damage responses, (2) Determine the effect of sustained ubiquitin chain formation on DNA damage responses, (3) Identify functionally important interacting proteins of alternative ubiquitin-linkages within DNA damage foci. Study Design: At first, polyubiquitin conjugates will be purified from cells treated by DNA damaging agents and ubiquitin conjugates will be assessed for their free ubiquitin chain content. Then, the effect of a conjugation- competent but deconjugation-deficient mutant of ubiquitin that I have recently developed will be tested to initiate a DNA repair response mechanism in the absence of DNA damage. Additionally, the effect of the mutant in making ubiquitin chains of a defined linkage will be tested under DNA damaging conditions to see if sustained ubiquitin chain formation sensitizes cells to DNA damage or inhibits the response. Finally, ubiquitin chain binding proteins will be purified by affinity purification from DNA damage induced cells to identify novel interacting proteins of alternative ubiquitin chains involved in DNA damage by mass spectrometry.