Structure and Mechanism in DNA Excision Repair

DNA切除修复的结构和机制

• 批准号: 7743501
• 负责人: GREGORY Lawrence VERDINE
• 金额: $ 36.31万
• 依托单位: HARVARD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-04-01 至 2013-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7743501
• 关键词: 3-methyladenine-DNA glycosylase; 7-methylguanine; 8-Oxoguanine DNA Glycosylase; Active Sites; Adenine; Area; Bacteria; Base Excision Repairs; Base Pairing; Biochemical; Cancer Etiology; Catalysis; Chemicals; Cleaved cell; Complex; DNA; DNA Damage; DNA Repair; DNA Repair Enzymes; DNA biosynthesis; DNA glycosylase; DNA repair protein; Disulfides; Engineering; Enzymes; Eukaryota; Event; Excision; Excision Repair; Genetic; Genome; Genome Scan; Goals; Human; Lesion; Lesion by Stage; Malignant Neoplasms; Molecular; Mutation; Nucleotide Excision Repair; Nucleotides; Pathway interactions; Physiological; Prokaryotic Cells; Proteins; Research; Roentgen Rays; Scanning; Series; Site; Solutions; Structure; Surgical incisions; System; Technology; Thymine Dimers; Time; Vertebral column; X-Ray Crystallography; adduct; analog; base; crosslink; endonuclease; interdisciplinary approach; mutant; nucleobase; nucleobase analog; prevent; programs; protein structure; public health relevance; repaired; research study; single molecule; tumorigenesis

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.

描述（由申请人提供）：对DNA的四个基础的自发损害是导致癌症突变的主要原因。这些遗传病变中的大多数通过两种途径中的任何一个，即碱基 - 脱孔DNA修复（BER）或核苷酸 - 脱离DNA修复（NER）校正。 BER主要是为了修复与DNA中正常核碱基相对较小的核苷酸酶的修复，而NER则修复了各种庞大的核核酶病变，例如胸腺素二聚体。 BER的关键成分是DNA糖基酶，专业的狩猎酶，它们扫描基因组以寻找特定种类的碱基损伤，然后从DNA骨架中催化损坏的碱基的切除。 NER没有如此专业的病变识别酶，而是使用两种蛋白质UVRA和UVRB，可以合作寻找许多不同种类的受损核碱基，唯一常见的特征是它们是笨重的。我们研究的长期目标是了解这些酶在广泛的正常DNA中如何定位它们的特殊损害，以及它们如何催化损害损坏的修复。对DNA损伤识别和去除的全面，基本的理解代表了肿瘤发生难题的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系统的基础分解修复蛋白，该蛋白可以直接修复高度诱变的病变8-氧气（OXOG） - 真核生物中的HOGGG1酶，在原核酸中的MUTM中的MUTM，或者在氧化腺苷中的修复：由氧化腺苷的修复：繁忙的基本粉碎物均取代了繁殖的依从物。病变，由muty蛋白（人类中的HMYH）催化。我们还建议研究通过DNA（细菌中的ALKA蛋白和人类AAG中的ALKA蛋白）修复各种遗传毒性甲基化加合物的糖基酶的病变识别。在NER方面，我们将研究途径中的早期事件，导致UVRB在病变上加载并募集UVRB依赖性核酸内切酶UVRC，该核酸内切酶UVRC，该核酸内切酶UVRC在侧面侧翼的部位切割DNA骨架。在这里，我们概述了一种基于广泛的，跨学科的方法，该方法采用化学交联和合成，光活性核碱基类似物的使用来捕获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的X射线晶体学和单分子DNA跟踪研究。这些研究共同提供了一个全面的分子级框架，以理解两个非常重要但非常不同的策略，以寻求和破坏DNA中的遗传毒性病变。公共卫生相关性：对DNA的核苷酸酶的损害会导致突变，并导致突变引起癌症。 DNA修复蛋白的责任是通过从基因组中消除受损的核碱基在DNA复制之前释放其诱变潜力来预防突变。在分子水平上，这种酶如何找到各种各样的病变和催化去除的方法。拟议计划的目的是填补大量缺失的癌症难题。