Structural and Mechanistic Studies of Error-Prone Polymerases

易错聚合酶的结构和机制研究

• 批准号: 7533198
• 负责人: Janice D Pata
• 金额: $ 25.84万
• 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 2013-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7533198
• 关键词: 2' -deoxycytidine 5' -triphosphate; 8-Oxo-2' -Deoxyguanosine; Address; Anti-Bacterial Agents; Antibiotic Resistance; Bacteria; Binding; Biochemical; Bypass; C-terminal; Cancerous; Cells; Characteristics; Class; Complex; Computing Methodologies; DNA; DNA Damage; DNA Replication Damage; DNA biosynthesis; DNA chemical synthesis; 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; Numbers; PCNA gene; Pharmaceutical Preparations; Polymerase; Positioning Attribute; Process; Property; Protein S; Proteins; Public Health; Rate; 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; repaired

DESCRIPTION (provided by applicant): 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. 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.

描述（由申请人提供）：Y-家庭DNA聚合酶通过允许复制继续在DNA模板中的相反病变来帮助细胞耐受DNA损伤。这种转移DNA合成可以准确，保留遗传信息的完整性，或者可能容易出错，即使模板链中的DNA损伤稍后修复，也会在基因组中产生突变。属于Dinb亚家族的Y家庭聚合酶能够通过在病变与病变相对的脱氧胞苷核苷酸掺入脱氧胞苷核苷酸来准确绕过受损的脱氧瓜氨酸碱基。与其他类型的Y-家庭聚合酶相比，DINB酶通常会造成的基本替代错误，但它们会产生单基删除突变，在复制过程中，在复制过程中跳过了模板基碱，以高速率跳过。我们将硫酸酸性的Dinb同源物（DBH）用作DNA聚合酶的Dinb类别的模型。已证明DBH可以准确有效地绕过脱氧鸟苷碱的DNA损伤。它表现出强烈的偏爱，即使在未损坏的DNA上也掺入了脱氧胞苷核苷酸。并且它以特定序列以异常高的速率生成单基础缺失误差。该提案的目的是对各种Y家庭DNA聚合酶之间的结构差异如何产生不同的病变 - 型 - - 型 - 型活性和DNA复制保真度。我们的核心假设是，DBH的夸张的突变特异性和病变 - 型 - - 型活性将使我们更容易地识别影响这些活动的结构特征。具体目的是（1）确定DBH如何生成单基础缺失突变，（2）以阐明DBH用于复制受损DNA的机制，以及（3）表征DBH如何受到与其他蛋白质相互作用的调节。我们将结合X射线晶体学，计算和生化方法来解决这些问题。这些研究将有助于我们理解Y-家庭聚合酶如何帮助细胞耐受DNA损伤以及它们如何将突变引入基因组。公共卫生相关性：人类细胞中多个突变的积累会导致癌细胞的生长，而细菌中的突变会导致抗生素耐药性。 Y-家庭DNA聚合酶似乎是原核生物和真核细胞中许多突变的原因。在适当的时候抑制这些聚合酶可能是防止癌症进展或增加抗菌药物治疗功效的有用方法。