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在相反方向继续，帮助细胞耐受DNA损伤。 DNA模板中的损伤这种跨损伤DNA合成可以是准确的，保留了细胞的完整性。 基因信息，或者它可以是容易出错的，即使基因组中的DNA损伤，也会在基因组中产生突变。 随后修复模板链。属于DinB亚家族的Y家族聚合酶能够 通过掺入脱氧胞苷核苷酸，准确地绕过受损的脱氧鸟苷碱基， 损伤。DinB酶通常比其他类型的Y家族产生更少的碱基替换错误 聚合酶，但他们使单碱基缺失突变，其中模板碱基被跳过， 复制，以高速率。我们使用来自酸热硫化叶菌的DinB同系物（Dbh）作为模型 DinB类DNA聚合酶。Dbh已被证明可以准确有效地绕过DNA 脱氧鸟苷碱基的损伤;它显示出对掺入脱氧胞苷核苷酸的强烈偏好 即使在未受损的DNA上也是如此;并且它在特定的条件下以异常高的速率产生单碱基缺失错误， 序列的本建议的目的是提供一个更完整的了解如何结构 各种Y家族DNA聚合酶之间的差异引起不同的病变旁路活性和DNA聚合酶活性。 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 Dbh的活性将使我们更容易识别影响这些活性的结构特征。的 具体的目的是（1）确定Dbh如何产生单碱基缺失突变，（2）阐明Dbh基因的突变机制。 Dbh用于复制受损DNA的机制，以及（3）表征Dbh如何通过相互作用进行调节 与其他蛋白质。我们将综合运用X射线晶体学计算学和生物化学 解决这些问题的方法。这些研究将有助于我们理解Y家族如何 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。公共卫生相关性声明 人类细胞中多个突变的积累可导致癌细胞生长，而人类细胞中的突变可导致癌细胞生长。 细菌会导致抗生素耐药性。Y家族DNA聚合酶似乎是许多 在原核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，以防止癌症的进展或提高疗效， 抗菌药物治疗。