FIDELLITY OF HUMAN POLYMERASE EPSILON

人类聚合酶 Epsilon 的保真度

• 批准号: 8360726
• 负责人: Zachary F Pursell
• 金额: $ 7.1万
• 依托单位: TULANE UNIVERSITY OF LOUISIANA
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-17 至 2012-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8360726
• 关键词: Alleles; Animal Model; Cell Line; Cells; DNA biosynthesis; DNA-Directed DNA Polymerase; Eukaryota; Exonuclease; Funding; Genetic Programming; Genome; Genome Stability; Goals; Grant; Human; Human Cell Line; In Vitro; Knock-in Mouse; Lead; Mammalian Cell; Measures; Mentors; Mismatch Repair; Mus; Mutation; National Center for Research Resources; Pattern; Play; Polymerase; Principal Investigator; Process; Recombinant adeno-associated virus (rAAV); Research; Research Infrastructure; Resources; Role; Source; System; Technology; Tumor-Derived; United States National Institutes of Health; Work; Yeasts; cancer cell; cancer genetics; cost; genome-wide; in vivo; mutant; tumorigenesis

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Spontaneous mutation rates in normal somatic mammalian cells are such that less than one mutation occurs per genome duplication. By contrast, cancer cells are characterized by having unstable genomes and recent genome-wide sequencing of tumor-derived cell lines has revealed thousands of mutations throughout their genome. DNA replication is a normally highly faithful process that plays an essential role in maintaining low spontaneous mutation rates. Three DNA polymerases (Pols), Pols a, d and e, are responsible for the vast majority of replicative DNA synthesis in eukaryotes. Pols d and e have been studied in model organisms and are highly accurate due to having a high replication fidelity combined with an intrinsic proofreading exonuclease activity. Mutations in yeast and mouse Pol e that reduce this high replication fidelity lead to increases in mutation rates and tumorigenesis. The contributions to genome stability and replication fidelity in human cells are currently unknown for human Pol e. The goal of this project is to develop human cell lines with the endogenous copies of Pol e changed to residues demonstrated to reduce in vitro replication fidelity. We will employ existing recombinant adeno-associated virus technology to generate knock-in alleles of human Pol e in mismatch repair-deficient and matched mismatch repair-corrected human cell lines. We will first characterize mutation rates and then determine patterns of mutations by sequencing the HPRT locus. We are currently working on characterizing the in vitro replication fidelity of human Pol e. Additionally, we are developing a system to express and purify mutants of Pol e and characterize the changes to in vitro replication fidelity caused by these mutator mutations. Combined with the results from this project, we hope to correlate changes to mutation rates and patterns caused by mutator mutants in vivo to the pattern of mutations made by those same mutants in vivo and measure directly the contribution of human Pol e to genome stability.

该子项目是利用资源的众多研究子项目之一 由 NIH/NCRR 资助的中心拨款提供。子项目的主要支持 并且子项目的主要研究者可能是由其他来源提供的， 包括其他 NIH 来源。 子项目可能列出的总成本 代表子项目使用的中心基础设施的估计数量， NCRR 赠款不直接向子项目或子项目工作人员提供资金。 正常哺乳动物体细胞的自发突变率使得每个基因组重复发生的突变少于一个。相比之下，癌细胞的特点是基因组不稳定，最近对肿瘤来源的细胞系进行全基因组测序揭示了其基因组中存在数千个突变。 DNA复制通常是一个高度忠实的过程，在维持低自发突变率方面发挥着重要作用。三种 DNA 聚合酶 (Pols)，Pols a、d 和 e，负责真核生物中绝大多数复制性 DNA 合成。 Pols d 和 e 已在模型生物体中进行了研究，并且由于具有高复制保真度和内在的校对核酸外切酶活性，因此高度准确。酵母和小鼠 Pol e 的突变降低了这种高复制保真度，导致突变率和肿瘤发生增加。目前尚不清楚人类 Pol e 对人类细胞中基因组稳定性和复制保真度的贡献。 该项目的目标是开发人类细胞系，将 Pol e 的内源副本更改为经证明可降低体外复制保真度的残基。我们将利用现有的重组腺相关病毒技术，在错配修复缺陷和匹配的错配修复校正的人类细胞系中产生人类 Pol e 的敲入等位基因。我们将首先表征突变率，然后通过对 HPRT 位点进行测序来确定突变模式。 我们目前正致力于表征人类 Pol e 的体外复制保真度。此外，我们正在开发一个系统来表达和纯化 Pol e 突变体，并表征由这些突变体突变引起的体外复制保真度的变化。结合该项目的结果，我们希望将体内增变突变体引起的突变率和模式的变化与这些相同突变体在体内产生的突变模式相关联，并直接测量人类 Pol e 对基因组稳定性的贡献。