Accessory Factors in DNA Repair

碱基切除修复(BER)是修复大多数碱基损伤的途径，包括烷化碱基、氧化碱基和无嘌呤/脱嘧啶(AP)位点。该途径是由识别和移除DNA中特定类型的改变的碱基的许多DNA糖基酶中的一种启动的。我们最近已经证明，BER效率可以通过控制辅助因子的表达来调节。具体地说，我们在体外和体内证明了CUX1蛋白的切割重复结构域刺激了8-氧鸟嘌呤DNA糖基酶OGG1的两种酶活性：去除错误碱基的DNA糖基酶活性和导致单链断裂的AP/裂解酶活性。与这些发现一致的是，CUX1基因敲除延迟了8-氧鸟嘌呤损伤的修复，而CUX1的过度表达则加速了8-氧鸟嘌呤损伤的修复。这些结果和其他几个小组的结果开辟了一个新的研究领域：DNA糖基酶不是单独发挥作用的，而是可以被辅助因子刺激的。*我建议确定针对烷化或氧化碱基的特定DNA糖基酶的全部辅助因子。我们将从OGG1开始，并将我们的探索依次扩展到其他3种针对氧化碱基的DNA糖基酶(NEIL1、NEIL2和NTH1)和修复烷化碱基的DNA糖基酶MPG。*我们假设，包含切割重复结构域的大多数蛋白质都可以与OGG1相互作用，并刺激OGG1。在目标1中，我们将研究每个切割重复蛋白作为OGG1辅助因子的能力。在我们的体外DNA修复实验中，由于其他DNA糖基酶不受切割重复序列的刺激，我们假设这些其他酶受到有待确认的辅助因素的刺激。在目标2中，我将描述三种互补的方法来确定DNA糖基酶的蛋白质伙伴和影响误码率效率的蛋白质。一种方法将采用亲和纯化方法，将DNA糖基酶与链球菌标签II肽融合为诱饵；第二种方法称为BioID，将使用包含突变的生物素连接酶的融合蛋白，该酶将使相互作用的蛋白质和相邻蛋白生物素化。质谱分析将鉴定纯化的蛋白质和生物素化的蛋白质。第三种方法将涉及使用带有荧光报告的DNA修复试验来筛选siRNA。在目标3中，将使用活细胞显微镜、蛋白质-蛋白质相互作用分析、DNA修复分析和辐射/耐药分析来验证特定蛋白质在BER中的意义。*这项工作将极大地提高我们对这一基本DNA修复途径的机制的理解。所获得的知识可能会提出新的策略，在神经退行性疾病的情况下刺激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。

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.**