FIDELLITY OF HUMAN POLYMERASE EPSILON

人类聚合酶 Epsilon 的保真度

• 批准号: 8168375
• 负责人: Zachary F Pursell
• 金额: $ 8.88万
• 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 2011-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8168375
• 关键词: Alleles; Animal Model; Cell Line; Cells; Computer Retrieval of Information on Scientific Projects Database; DNA biosynthesis; DNA-Directed DNA Polymerase; Eukaryota; Exonuclease; Funding; Genome; Genome Stability; Goals; Grant; Human; Human Cell Line; In Vitro; Institution; Knock-in Mouse; Lead; Mammalian Cell; Measures; Mismatch Repair; Mus; Mutation; Pattern; Play; Polymerase; Process; Recombinant adeno-associated virus (rAAV); Research; Research Personnel; Resources; Role; Source; System; Technology; Tumor-Derived; United States National Institutes of Health; Work; Yeasts; cancer cell; 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. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. 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.

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 正常哺乳动物体细胞中的自发突变率是这样的，即每次基因组复制发生少于一个突变。相比之下，癌细胞的特征在于具有不稳定的基因组，并且最近对肿瘤来源的细胞系的全基因组测序已经揭示了贯穿其基因组的数千个突变。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起着至关重要的作用。三种DNA聚合酶（Pos），Pos a、d和e，负责真核生物中绝大多数的复制性DNA合成。已经在模式生物中研究了聚合物d和e，并且由于具有高复制保真度与内在校正外切核酸酶活性的组合，聚合物d和e是高度准确的。酵母和小鼠Pol e中降低这种高复制保真度的突变导致突变率和肿瘤发生的增加。目前尚不清楚人Pol e对人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细胞系，其中Pol e的内源性拷贝被改变为被证明降低体外复制保真度的残基。我们将采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细胞系中产生人Pol e的敲入等位基因。我们将首先描述突变率，然后通过对HPRT基因座进行测序来确定突变模式。 我们目前正致力于表征人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，并表征由这些增变因子突变引起的体外复制保真度的变化。结合本项目的结果，我们希望将体内增变因子突变体引起的突变率和模式的变化与体内相同突变体引起的突变模式相关联，并直接测量人类Pol e对基因组稳定性的贡献。