BYPASS FIDELITY OF OXIDATIVE DAMAGE LESIONS BY Y-FAMILY DNA POLYMERASE

Y 家族 DNA 聚合酶绕过氧化损伤损伤的保真度

• 批准号: 7955159
• 负责人: DINSHAW J PATEL
• 金额: $ 0.73万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-04-01 至 2010-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7955159
• 关键词: 4-nitroimidazole; Active Sites; Adopted; Aging; Amino Acids; Architecture; Base Pairing; Bypass; Cardiovascular Diseases; Cells; Computer Retrieval of Information on Scientific Projects Database; Cytosine; DNA; DNA Damage; DNA biosynthesis; DNA-Directed DNA Polymerase; Event; Family; Funding; Genomics; Goals; Grant; Infection; Inflammation; Institution; Lesion; Link; Malignant Neoplasms; Molecular; Molecular Conformation; Mutation; Neurodegenerative Disorders; Oxidative Stress; Polymerase; Reactive Oxygen Species; Recruitment Activity; Research; Research Personnel; Resources; Risk; Shapes; Site; Source; United States National Institutes of Health; adduct; base; oxidative damage; response; spiroiminodihydantoin; structural biology

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Genomic integrity depends critically on the fidelity and efficiency of DNA replication. High-fidelity DNA polymerases that replicate genomic DNA can stall on certain DNA damage sites, and one or more lesion-bypass Y-family polymerases are recruited to transit the lesion. Such bypass polymerases have a higher error rate and lower processivity on undamaged DNA templates, but can insert a base opposite a lesion site and extend from a damaged base pair in error-free (mutation-avoiding) or error-prone (mutation-generating) manner. Our goal is to understand the molecular mechanisms that define the mutagenic events associated with replication of oxidative damage lesions by bypass polymerases. An increased risk for developing cancer has been linked to oxidative stress due to the overproduction of reactive oxygen species resulting from the response of cells to inflammation and infection. Elevated levels of oxidative damage lesions in genomic DNA have also been associated with neurodegenerative diseases, aging, and cardiovascular disorders. Our crystallographic studies are undertaken on the most prevalent oxidative damage lesions, namely, 8-oxoguanine, the stable ring-opened 5-guanidino-4-nitroimidazole adduct and the fused bicyclic spiroiminodihydantoin adduct, placed in the context of DNA template strands of the active site of Dpo4 Y-family polymerase. Previously, our group uncovered structural and functional features that enable low-fidelity Dpo4 polymerase to achieve predominantly error-free insertion of a cytosine base opposite the 8-oxoguanine (oxoG) lesion. We found of The oxoG lesion adopts an anti conformation within the Dpo4 active site, that is necessary for pairing with dCTP, with the recognition event facilitated by multiple and favorable contacts of Dpo4 amino acid residues with the oxoG. We have also demonstrated that Dpo4 undergoes stepwise translocations throughout the catalytic cycle that are distinct from the corresponding translocation events observed in high-fidelity polymerases. Our efforts should elucidate the common factors that promote error-free or error-prone DNA synthesis opposite and past the oxidative damage lesions of varying size and shape, as well as lesion architecture and preferred conformations.

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 基因组的完整性关键取决于DNA复制的保真度和效率。复制基因组DNA的高保真DNA聚合酶可以在某些DNA损伤位点上停滞，并且一个或多个病变旁路Y家族聚合酶被募集以通过病变。这种旁路聚合酶在未受损的DNA模板上具有较高的错误率和较低的持续合成能力，但可以在损伤位点对面插入碱基，并以无错误（避免突变）或易出错（产生突变）的方式从受损碱基对延伸。 我们的目标是了解定义与旁路聚合酶复制氧化损伤病变相关的致突变事件的分子机制。患癌症的风险增加与氧化应激有关，氧化应激是由于细胞对炎症和感染的反应导致活性氧的过度产生。基因组DNA中氧化损伤病变水平升高也与神经退行性疾病、衰老和心血管疾病相关。 我们的晶体学研究进行最普遍的氧化损伤病变，即8-氧代鸟嘌呤，稳定的开环5-胍基-4-硝基咪唑加合物和稠合双环螺亚氨基二乙内酰脲加合物，放置在DPO 4 Y-家族聚合酶的活性位点的DNA模板链的上下文中。 以前，我们的研究小组发现了结构和功能特征，使低保真度Dpo 4聚合酶能够实现与8-氧代鸟嘌呤（oxoG）病变相对的胞嘧啶碱基的主要无错误插入。我们发现的oxoG病变采用的Dpo 4活性位点内的反构象，这是必要的配对与dCTP，与识别事件的Dpo 4氨基酸残基与oxoG的多个和有利的接触促进。 我们还表明，DPO 4经历了逐步易位在整个催化循环中观察到的高保真聚合酶中的相应的易位事件是不同的。我们的努力应该阐明的共同因素，促进错误或错误倾向的DNA合成相反和过去的氧化损伤病变的不同大小和形状，以及病变的架构和首选的构象。