Repair Mechanisms For Oxidative DNA Damage

DNA 氧化损伤的修复机制

• 批准号: 7592049
• 负责人: David M Wilson
• 金额: $ 74.94万
• 依托单位: NATIONAL INSTITUTE ON AGING
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7592049
• 关键词: 8-oxoguanine; APTX gene; ATP phosphohydrolase; Aging; Base Excision Repairs; Biochemical; C-terminal; Cell Aging; Cell Death; Cell Respiration; Cell Survival; Cell physiology; Cells; Chromosomal Stability; Chronic; Cleaved cell; Cockayne Syndrome; Complex; Coupled; Cultured Cells; Cutaneous; DNA; DNA Damage; DNA Polymerase beta; DNA Repair; DNA Structure; DNA strand break; Defect; Disease; Disease susceptibility; Dominant-Negative Mutation; Enzymes; Failure; Functional disorder; Future; Genetic; Genetic Transcription; Growth; Human; Incidence; Individual; Interphase Cell; Investigation; Laboratories; Ligation; Link; Lipids; Malignant Neoplasms; Mechanics; Mediating; Methods; Modeling; Molecular; Mus; Nerve Degeneration; Neurodegenerative Disorders; Neurologic; Neurons; Nucleic Acids; Numbers; PCNA gene; Pathology; Pathway interactions; Photosensitivity; Population; Premature aging syndrome; Process; Property; Proteins; RNA; RNA Interference; Range; Reactive Oxygen Species; Role; SWI2/SNF2; Scaffolding Protein; Single Strand Break Repair; Site; Specificity; Spinocerebellar Ataxias; Structure; Surgical incisions; Symptoms; System; Telomere Maintenance; Testing; Therapeutic; Therapeutic Agents; Thinking; Variant; Work; XRCC1 gene; age related; cancer cell; cancer therapy; cell age; chemotherapeutic agent; chromatin remodeling; cytotoxic; design; disorder risk; helicase; homologous recombination; novel; oxidative DNA damage; protein function; protein protein interaction; repaired; response; scaffold; tyrosyl-DNA phosphodiesterase

Much of our effort has been to define the structure-function mechanisms and biochemical properties of Ape1, the major protein for repairing abasic sites and certain 3-damages in DNA. Our work has revealed that Ape1 cleaves at AP sites in single-stranded regions of complex, biologically-relevant DNA structures, such as bubble and fork intermediates. These findings expand the known repertoire of substrates processed by this enzyme, and suggest novel functions for Ape1 likely coupled to transcription and/or replication. We recently found that the protein defective in the human segmental progeroid, Cockayne Syndrome B (CSB), physically and functionally interacts with Ape1. We are now determining the precise molecular contributions of CSB to BER and testing more exhaustively for possible helicase- and remodeling-type activities for CSB on a variety of DNA and RNA substrates. These studies in total will determine the molecular functions of CSB and how certain activities contribute to the associated disease manifestation. Last, we are designing methods to strategically regulate Ape1 repair activity using a dominant-negative approach in the hopes of developing more effective anti-cancer treatment paradigms. In addition to the investigations above, we are elucidating the biochemical and cellular contributions of XRCC1, a major SSB repair (SSBR) factor. This protein functions primarily as a scaffold component, orchestrating specific protein-protein interactions required for efficient DNA repair. Our studies (i) suggest a link of XRCC1 to replication via an interaction with PCNA, (ii) argue against a role for XRCC1 in the early steps of BER, and (iii) indicate a biologically-relevant role for its interaction with DNA polymerase beta and in the subsequent repair step, nick ligation. Recent work has identified associations of XRCC1 with proteins defective in human neurodegenerative disorders AOA1 (Aprataxin) and SCAN1 (TDP1). We are assessing the role of XRCC1 in non-dividing (neuronal) cells and age-related pathologies (namely neurodegeneration) using cell culture models and heterozygous mice. The contributions of XRCC1 to DNA damage responses, chromosome stability, and telomere maintenance are concurrently being evaluated in dividing human cells using chronic RNAi knockdown strategies. The ongoing investigations will delineate the contribution of SSBs (i.e. an XRCC1 deficiency), a common DNA intermediate, to cancer promotion (genetic stability) and neurodegenerative disease (neuronal cell viability).

我们的大部分工作都是为了确定APE1的结构-功能机制和生化特性，APE1是修复DNA中基本位点和某些3-损伤的主要蛋白质。我们的工作揭示了APE1在复杂的、生物相关的DNA结构的单链区域的AP位点上裂解，如气泡和叉状中间体。这些发现扩大了已知的由这种酶处理的底物的谱系，并表明APE1可能与转录和/或复制有关的新功能。我们最近发现，人类节段性孕激素的缺陷蛋白，Cockayne综合征B(CSB)，在物理和功能上与APE1相互作用。我们现在正在确定CSB对误码率的精确分子贡献，并更详尽地测试CSB在各种DNA和RNA底物上可能的解旋酶和重塑类型的活性。总体而言，这些研究将确定CSB的分子功能以及某些活动如何有助于相关疾病的表现。最后，我们正在设计方法，使用显性-负性方法对APE1修复活动进行战略性调节，以期开发出更有效的抗癌治疗范例。 除了上述研究外，我们还在阐明XRCC1的生化和细胞作用，XRCC1是一种主要的SSB修复(SSBR)因子。这种蛋白质主要作为支架成分，协调有效DNA修复所需的特定蛋白质-蛋白质相互作用。我们的研究(I)表明XRCC1通过与增殖细胞核抗原的相互作用与复制有关，(Ii)反对XRCC1在BER的早期步骤中的作用，以及(Iii)表明XRCC1与DNA聚合酶β的相互作用以及在随后的修复步骤--缺口连接中的生物相关作用。最近的工作已经确定了XRCC1与人类神经退行性疾病AOA1(Aprataxin)和SCAN1(Tdp1)缺陷蛋白的相关性。我们正在使用细胞培养模型和杂合子小鼠评估XRCC1在未分裂(神经元)细胞和与年龄相关的病理(即神经退行性变)中的作用。在使用慢性RNAi敲除策略分裂人类细胞时，正在同时评估XRCC1对DNA损伤反应、染色体稳定性和端粒维持的贡献。正在进行的研究将描绘SSB(即XRCC1缺乏)，一种常见的DNA中间体，对癌症促进(遗传稳定性)和神经退行性疾病(神经细胞活性)的贡献。