Accessory Factors in DNA Repair

DNA 修复中的辅助因素

• 批准号: RGPIN-2016-05155
• 负责人: Nepveu, Alain
• 金额: $ 2.77万
• 依托单位: McGill University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2019
• 资助国家: 加拿大
• 起止时间: 2019-01-01 至 2020-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=687057
• 关键词: Accessory; Factors; DNA; Repair

Base excision repair (BER) is the pathway that repairs most base lesions including alkylated bases, oxidized bases and apurinic/apyrimidinic (AP) sites. The pathway is initiated by one of many DNA glycosylases that recognize and remove specific types of altered bases in DNA. We have recently demonstrated that BER efficiency can be modulated by controlling the expression of an accessory factor. Specifically, we showed in vitro and in vivo that the Cut repeat domains from the CUX1 protein stimulate two enzymatic activities of the 8-oxoguanine DNA glycosylase, OGG1: the DNA glycosylase activity that removes the faulty base and the AP/lyase activity that introduces a single-strand break. In agreement with these findings, CUX1 knockdown delays, whereas CUX1 overexpression accelerates, the repair of 8-oxoguanine lesions. These results and that of a few other groups have opened a new area of investigation: DNA glycosylases do not function in isolation but can be stimulated by accessory factors.*** *I propose to identify the full repertoire of accessory factors for specific DNA glycosylases that target alkylated or oxidized bases. We will begin with OGG1 and extend our quest successively to the 3 other DNA glycosylases specific for oxidized bases (NEIL1, NEIL2, and NTH1) and the DNA glycosylase that repairs alkylated bases, MPG.*** *We hypothesize that most, if not all, proteins that contain a Cut repeat domain can interact with, and stimulate, OGG1. In aim 1, we will investigate the capacity of each Cut repeat protein to function as an accessory factor for OGG1. As other DNA glycosylases were not stimulated by Cut repeat in our in vitro DNA repair assay, we hypothesize that these other enzymes are stimulated by accessory factors that remain to be identified. In aim 2, I will describe three complementary approaches to identify protein partners of DNA glycosylases and proteins that impact on BER efficiency. One approach will employ affinity purification using as baits the DNA glycosylases fused to the strep-tag II peptide; a second approach, called BioID, will use fusion proteins containing a mutant biotin ligase that will biotinylate interacting proteins and neighbours. Mass spectrometry analysis will identify the purified proteins and the biotinylated proteins. A third approach will involve siRNA screen using a DNA repair assay with a fluorescent reporter. In aim 3, the implication of specific proteins in BER will be validated using live-cell microscopy, protein-protein interaction assays, DNA repair assays and radiation/drug resistance assays.*** This work will greatly improve our mechanistic understanding of this essential DNA repair pathway. The acquired knowledge may suggest novel strategies to stimulate DNA repair in the case of neurodegenerative diseases or alternatively, to reduce DNA repair efficiency in order to sensitize cancer cells to treatments causing DNA damage.**

碱基切除修复（BER）是修复大多数碱基损伤的途径，包括烷基化碱基、氧化碱基和脱嘌呤/脱嘧啶（AP）位点。该途径由许多DNA糖基化酶中的一种启动，这些酶识别并去除DNA中特定类型的改变的碱基。我们最近已经证明，BER效率可以调制通过控制的表达的辅助因素。具体而言，我们在体外和体内表明，CUX 1蛋白的切割重复结构域刺激8-氧代鸟嘌呤DNA糖基化酶OGG 1的两种酶活性：去除错误碱基的DNA糖基化酶活性和引入单链断裂的AP/裂解酶活性。与这些发现一致，CUX 1敲低延迟，而CUX 1过表达加速，8-氧代鸟嘌呤病变的修复。这些结果和其他一些研究小组的结果开辟了一个新的研究领域：DNA糖基化酶在孤立的情况下不起作用，但可以被辅助因子刺激。 * 我建议确定针对烷基化或氧化碱基的特定DNA糖基化酶的全部辅助因子。我们将从OGG 1开始开始，并将我们的探索依次扩展到对氧化碱基特异的其他3种DNA糖基化酶（NEIL 1，NEIL 2和NTH 1）和修复烷基化碱基的DNA糖基化酶MPG。 * 我们假设大多数（如果不是全部）含有切割重复结构域的蛋白质可以与OGG 1相互作用并刺激OGG 1。在目标1中，我们将研究每个切割重复蛋白作为OGG 1的辅助因子发挥作用的能力。由于其他DNA糖基化酶在我们的体外DNA修复试验中没有受到切割重复序列的刺激，我们假设这些其他酶受到有待鉴定的辅助因子的刺激。在目标2中，我将描述三种互补的方法来鉴定DNA糖基化酶和影响BER效率的蛋白质的蛋白质伴侣。一种方法将采用亲和纯化，使用与strep标签II肽融合的DNA糖基化酶作为诱饵;第二种方法称为BioID，将使用含有突变生物素连接酶的融合蛋白，该突变生物素连接酶将生物素化相互作用的蛋白质和邻居。质谱分析将鉴定纯化的蛋白和生物素化的蛋白。第三种方法将涉及使用具有荧光报告基因的DNA修复测定进行siRNA筛选。在目标3中，将使用活细胞显微镜、蛋白质-蛋白质相互作用测定、DNA修复测定和辐射/耐药性测定来验证BER中特定蛋白质的含义。 这项工作将大大提高我们对这一重要DNA修复途径的机械理解。所获得的知识可能会提出新的策略，以刺激神经退行性疾病的DNA修复，或者降低DNA修复效率，以使癌细胞对导致DNA损伤的治疗敏感。