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.

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