Structural and Mechanistic Studies of Error-Prone Polymerases

易错聚合酶的结构和机制研究

• 批准号: 8312533
• 负责人: Janice D Pata
• 金额: $ 26.56万
• 依托单位: WADSWORTH CENTER
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-08-01 至 2014-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8312533
• 关键词: Address; Anti-Bacterial Agents; Antibiotic Resistance; Bacteria; Binding; Biochemical; Bypass; C-terminal; Cancerous; Cells; Characteristics; Complex; Computing Methodologies; DNA; DNA Damage; DNA Replication Damage; DNA biosynthesis; DNA lesion; DNA-Directed DNA Polymerase; Deletion Mutation; Deoxycytidine; Deoxyguanosine; Enzymes; Eukaryotic Cell; Family; Foundations; Genetic; Genome; Goals; Homologous Gene; Human; Investigation; Knowledge; Lead; Lesion; Lung; Malignant Neoplasms; Methods; Modeling; Mutagenesis; Mutation; Nucleotides; Pharmaceutical Preparations; Polymerase; Positioning Attribute; Process; Prokaryotic Cells; Property; Protein S; Proteins; Relative (related person); Roentgen Rays; Role; Specificity; Staging; Structure; Sulfolobus acidocaldarius; Sulfolobus solfataricus; Testing; Time; Tobacco smoke; Variant; adduct; base; cancer cell; cell growth; preference; prevent; public health relevance; repaired

Project Summary The Y-family DNA polymerases help cells tolerate DNA damage by allowing replication to continue opposite lesions in the DNA template. This translesion DNA synthesis can be accurate, preserving the integrity of the genetic information, or it can be error-prone, producing a mutation in the genome even if the DNA damage in the template strand is repaired later. The Y-family polymerases that belong to the DinB subfamily are able to bypass damaged deoxyguanosine bases accurately by incorporating deoxycytidine nucleotides opposite the lesion. The DinB enzymes generally make fewer base-substitution errors than other types of Y-family polymerases, yet they make single-base deletion mutations, where a template base is skipped during replication, at a high rate. We are using the DinB homolog (Dbh) from Sulfolobus acidocaldarius as a model for the DinB class of DNA polymerases. Dbh has been demonstrated to accurately and efficiently bypass DNA damage at deoxyguanosine bases; it displays a strong preference for incorporating deoxycytidine nucleotides even on undamaged DNA; and it generates single-base deletion errors at an exceptionally high rate at specific sequences. The objective of this proposal is to provide a more complete understanding of how structural differences among the various Y-family DNA polymerases give rise to differing lesion-bypass activity and DNA replication fidelity. Our central hypothesis is that the exaggerated mutational specificity and lesion-bypass activity of Dbh will allow us to more easily identify the structural features that influence these activities. The specific aims are (1) to determine how Dbh generates single-base deletion mutations, (2) to elucidate the mechanisms Dbh uses to replicate damaged DNA, and (3) to characterize how Dbh is regulated by interactions with other proteins. We will use a combination of X-ray crystallographic, computational and biochemical approaches to address these issues. These studies will contribute to our understanding of how the Y-family polymerases help cells tolerate DNA damage and also how they introduce mutations into the genome. Statement of Public Health Relevance An accumulation of multiple mutations in human cells can lead to cancerous cell growth, while mutations in bacteria can lead to antibiotic resistance. The Y-family DNA polymerases appear to be responsible for many of the mutations produced in both prokaryotic and eukaryotic cells. Inhibiting these polymerases, at appropriate times, could be a useful way to prevent cancers from progressing or to increase the efficacy of antibacterial drug treatments.

项目摘要 Y家族DNA聚合酶通过允许复制继续相反的方式帮助细胞耐受DNA损伤 DNA模板中的损伤。这种跨病变的DNA合成可以是准确的，保留了 遗传信息，或者它可能容易出错，在基因组中产生突变，即使DNA损伤在 模板链随后被修复。属于DinB亚家族的Y家族聚合酶能够 准确地绕过受损的脱氧鸟苷碱基，方法是将脱氧胞苷与 损伤。与其他类型的Y家族相比，DinB酶通常较少发生碱基替换错误 聚合酶，但它们会造成单碱基缺失突变，在此过程中跳过模板碱基 复制，速度很快。我们使用来自Sulfolobus acidocaldarius的DinB同源基因(DBH)作为模型 用于DinB类DNA聚合酶。DBH已被证明可以准确有效地绕过DNA 损伤脱氧鸟苷碱基；它表现出强烈的结合脱氧胞苷核苷酸的偏好 甚至在未受损的DNA上也是如此；它在特定的特定情况下以极高的比率产生单碱基缺失错误 序列。这项建议的目标是提供一个更完整的理解如何 不同Y家族DNA聚合酶之间的差异导致不同的病变旁路活性和DNA 复制保真度。我们的中心假设是夸大的突变特异性和病变旁路 胸径活动将使我们能够更容易地确定影响这些活动的结构特征。这个 具体目标是(1)确定胸径如何产生单碱基缺失突变，(2)阐明 胸径用于复制受损DNA的机制，以及(3)描述胸径是如何通过相互作用来调节的 与其他蛋白质结合。我们将使用X射线结晶学、计算和生物化学的组合 解决这些问题的方法。这些研究将有助于我们理解Y家族是如何 聚合酶帮助细胞耐受DNA损伤，以及它们如何将突变引入基因组。《与公共卫生相关的声明》 人类细胞中多种突变的积累可以导致癌细胞的生长，而突变在 细菌可能会导致抗生素耐药性。Y家族DNA聚合酶似乎对许多 在原核细胞和真核细胞中产生的突变。抑制这些聚合酶，在 适当的时间，可能是一种有效的方法来防止癌症的进展或提高 抗菌药物治疗。