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，包括烷基化碱基、氧化碱基和无嘌呤/无嘧啶（AP）位点。该途径是由识别和去除DNA中特定类型的改变碱基的许多DNA糖基酶之一启动的。我们最近证明了误码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Cut repeat结构域刺激8-氧鸟嘌呤DNA糖基酶OGG1的两种酶活性：去除错误碱基的DNA糖基酶活性和引入单链断裂的AP/裂解酶活性。与这些发现一致，CUX1敲低延迟，而CUX1过表达加速8-氧鸟嘌呤损伤的修复。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糖基化酶不是孤立地起作用的，而是可以受到辅助因子的刺激。*** *我建议鉴定出针对烷基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氧化碱基特异性的其他3种DNA糖基酶（NEIL1， NEIL2和NTH1）和修复烷基化碱基的DNA糖基酶MPG。*** *我们假设大多数（如果不是全部的话）含有切割重复结构域的蛋白质可以与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辅助因子的功能。在我们的体外DNA修复实验中，由于其他DNA糖基酶没有被切割重复刺激，我们假设这些其他酶受到尚未确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识别DNA糖基酶的蛋白质伴侣和影响BER效率的蛋白质。一种方法将采用亲和纯化，使用融合到链球菌标签II肽的DNA糖基酶作为诱饵；第二种方法称为BioID，它将使用含有突变生物素连接酶的融合蛋白，这种融合酶将使相互作用的蛋白和邻近蛋白生物素化。质谱分析将鉴定纯化蛋白和生物素化蛋白。第三种方法将涉及使用带有荧光报告基因的DNA修复试验进行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基本DNA修复途径的机制理解。获得的知识可能为神经退行性疾病中刺激DNA修复的新策略提供建议，或者降低DNA修复效率以使癌细胞对引起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.**