Structure and Mechanism in DNA Excision Repair

描述（由申请人提供）：DNA四个碱基的自发损伤是导致癌症突变的主要原因。大多数这些遗传性损伤通过两种途径中的任一种来纠正，碱基切除DNA修复（BER）或核苷酸切除DNA修复（NER）。BER主要负责修复相对于DNA中的正常核碱基具有相对小的简单变化的核碱基，而NER修复各种各样的大的核碱基损伤，如胸腺嘧啶二聚体。BER的关键成分是DNA糖基化酶，这是一种专业的损伤搜索酶，它扫描基因组以寻找特定类型的碱基损伤，然后催化从DNA骨架中切除受损的碱基。NER没有这种专门的损伤识别酶，而是使用两种蛋白质，UvrA和UvrB，合作寻找许多不同种类的受损核碱基，唯一的共同特征是它们体积庞大。我们研究的长期目标是了解这些酶如何在广阔的正常DNA中定位它们特定类型的损伤，以及一旦定位后它们如何催化损伤的修复。对DNA损伤识别和去除的全面、基本的理解代表了肿瘤发生之谜的一个主要方面的解决方案。在拟议的研究中，我们将研究所谓的GO系统的碱基切除修复蛋白，该系统负责直接修复高致突变性损伤8-氧代鸟嘌呤（oxoG）-真核生物中的hOgg 1酶和原核生物中的MutM-或修复oxoG中的致突变性腺嘌呤：由MutY蛋白（人类中的hMYH）催化的未修复的oxoG损伤的错误复制引起的碱基对。我们还建议研究损伤识别的糖基化酶，修复各种遗传毒性的甲基化加合物的DNA（在细菌和Aag在人类的AlkA蛋白）。在NER方面，我们将研究导致UvrB加载到病变和招募UvrB依赖性内切核酸酶UvrC的途径中的早期事件，UvrC在病变两侧的位点切割DNA骨架。在这里，我们概述了一个基础广泛的，跨学科的方法，采用化学交联和合成，光敏核碱基类似物的使用，以陷阱BER和NER修复途径中的中间体。为了了解病变识别的动态方面，我们将采用时间分辨X射线晶体学和单分子DNA跟踪研究。总之，这些研究的目的是提供一个全面的分子水平的框架，了解两个非常重要的，但非常不同的策略，寻找和破坏DNA中的遗传毒性病变。公共卫生相关性：DNA核碱基的损伤导致突变，突变导致癌症。DNA修复蛋白的职责是在DNA复制释放其致突变潜力之前，通过从基因组中消除受损的核碱基来防止突变。在分子水平上，人们对这些酶如何定位它们不同的病变阵列并催化清除这些病变知之甚少;拟议计划的目标是填补癌症难题中缺失的那一大块。

DESCRIPTION (provided by applicant): Spontaneous damage to the four bases of DNA is a major cause of the mutations that give rise to cancer. Most of these genetic lesions are corrected by either of two pathways, base-excision DNA repair (BER) or nucleotide-excision DNA repair (NER). BER is primarily responsible for the repair of nucleobases having relatively small, simple changes with respect to the normal nucleobases in DNA, whereas NER repairs a wide variety of bulky nucleobase lesions such as thymine dimers. The key components of BER are DNA glycosylases, professional lesion-hunting enzymes that scan the genome in search of particular kinds of base damage, then catalyze excision of the damaged base from the DNA backbone. NER does not have such specialized lesion-recognition enzymes, but instead employs two proteins, UvrA and UvrB, to search cooperatively for damaged nucleobases of many different kinds, the only common feature being that they are bulky. The long-term goals of our studies are to understand how these enzymes locate their particular kinds of damage amidst the vast expanse of normal DNA, and how they catalyze repair of the damage once having located it. A comprehensive, fundamental understanding of DNA damage recognition and removal represents the solution to a major aspect of the tumorigenesis puzzle. In the proposed studies, we will study base-excision repair proteins of the so-called GO system that are responsible for either direct repair of the highly mutagenic lesion 8-oxoguanine (oxoG) - the hOgg1 enzyme in eukaryotes and MutM in prokaryotes - or repair of the mutagenic adenine in oxoG:A base-pair resulting from mis-replication of unrepaired oxoG lesions, catalyzed by the MutY protein (hMYH in humans). We also propose to investigate lesion recognition by glycosylases that repair a variety of genotoxic methylated adducts in DNA (the AlkA protein in bacteria and Aag in humans). On the NER front, we will study the early events in the pathway leading to the loading of UvrB onto a lesion and recruitment of a UvrB-dependent endonuclease, UvrC, which cleaves the DNA backbone at sites flanking the lesion. Here we outline a broad-based, interdisciplinary approach that employs the use of chemical crosslinking and synthetic, photoactive nucleobase analogs to trap intermediates in the BER and NER repair pathways. To understand dynamic aspects of lesion recognition, we will employ time-resolved X-ray crystallography and single-molecule DNA tracking studies. Together, these studies aim to provide a comprehensive molecular-level framework for understanding two very important but very different strategies for seeking out and destroying genotoxic lesions in DNA. PUBLIC HEALTH RELEVANCE: Damage to the nucleobases of DNA causes mutations, and mutations cause cancer. It is the responsibility of DNA repair proteins to prevent mutations by eradicating damaged nucleobases from the genome before DNA replication unleashes their mutagenic potential. How such enzymes locate their diverse array of lesions and catalyze removal is poorly understood at the molecular level; it is the goal of the proposed program to fill in that substantial missing piece of the cancer puzzle.