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家庭DNA聚合酶通过允许复制继续相反的细胞来帮助细胞耐受DNA损伤 DNA模板中的病变。这种转移DNA合成可以是准确的，可以保留 遗传信息，或者可能容易出错，即使在基因组中产生突变，即使DNA损伤 模板链稍后修复。属于Dinb亚家族的Y家庭聚合酶 绕过通过掺入对面的脱氧胞苷核苷酸来准确损坏了脱氧瓜烷碱基。 病变。与其他类型的Y家庭相比 聚合酶，但它们会产生单基删除突变，其中跳过了模板基础 复制，速度很高。我们正在使用硫酸酸性酸性的Dinb同源物（DBH）作为模型 用于DNA聚合酶的二氧化碳类。已证明DBH可以准确有效地绕过DNA 脱氧鸟氨酸碱基的损坏；它表现出强烈的偏爱融合脱氧胞苷核苷酸 即使在未损坏的DNA上；并且它以特定于特定的速率以异常高的速率生成单基数删除误差 序列。该提案的目的是对结构性的方式提供更完整的理解 各种Y家庭DNA聚合酶之间的差异会导致病变 - 型 - - 型活性和DNA不同 复制保真度。我们的中心假设是夸张的突变特异性和病变 - 巴巴 DBH的活动将使我们更容易地确定影响这些活动的结构特征。这 具体目的是（1）确定DBH如何生成单基础缺失突变，（2）阐明 DBH的机制用于复制受损的DNA，（3）表征DBH如何受到相互作用的调节 与其他蛋白质。我们将结合X射线晶体学，计算和生化 解决这些问题的方法。这些研究将有助于我们对Y家庭的理解 聚合酶有助于细胞耐受DNA损伤，并如何将突变引入基因组。公共卫生相关性声明 人类细胞中多重突变的积累会导致癌细胞的生长，而突变 细菌会导致抗生素耐药性。 Y家庭DNA聚合酶似乎是许多原因 在原核生物和真核细胞中产生的突变。抑制这些聚合酶 适当的时间，可能是防止癌症进步或提高疗效的有用方法 抗菌药物治疗。