Investigation of human DNA polymerase epsilon variants

人类 DNA 聚合酶 epsilon 变异体的研究

基本信息

批准号：
10220033
负责人：
Zachary F Pursell
金额：
$ 31.64万
依托单位：
TULANE UNIVERSITY OF LOUISIANA
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-08-15 至 2022-08-15
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10220033
关键词：
Address Affect Alleles Animal Model Automobile Driving Biochemical Bypass Cancer Etiology Cell Culture Techniques Cell Line Cell model Cells Collaborations Colorectal Neoplasms Complex DNA DNA Polymerase II DNA Repair DNA Sequence Alteration DNA biosynthesis DNA lesion DNA replication fork DNA-Directed DNA Polymerase Data Daughter Defect Development Disease Environment Enzymes Exhibits Exonuclease Future Genes Genetic Genetic Engineering Genome Genome Stability Genomic Instability Genomics Goals Health Holoenzymes Human Human Cell Line Human Engineering Impairment In Vitro Intervention Investigation Kinetics Knock-in Knowledge Laboratories Lead Lesion Link Malignant Neoplasms Measures Methods Mismatch Repair Missense Mutation Mission Mutagenesis Mutation Nonsense Mutation Normal Cell Nuclear Phenotype Phosphodiesterase I Physiological Point Mutation Polymerase Positioning Attribute Property Reading Research Research Design Ribonucleotides Somatic Mutation Testing Therapeutic Therapeutic Intervention United States National Institutes of Health Uterine Neoplasms Variant Work base burden of illness cancer cell cancer genome cancer type genomic locus human DNA immune checkpoint blockade in vitro activity in vivo innovation insight interdisciplinary approach mouse model mutant next generation sequencing novel polymerization prevent tumor tumorigenesis

项目摘要

SUMMARY Cancer cells typically exhibit genetic instability and accumulate thousands to hundreds of thousands of mutations in their genome. Replicative DNA polymerases are responsible for copying the vast majority of nuclear DNA. Conditions that reduce their ability to accurately and efficiently synthesize daughter DNA molecules can contribute directly to this increased mutagenesis. Recently, heterozygous missense mutations were identified in the exonuclease domain of DNA Polymerase (Pol) ε from several tumor types. These tumors contain the highest number of somatic mutations identified in tumor genomes to date. Despite these significant consequences, the mechanisms that drive this mutagenesis and how this ultimately affects tumorigenesis remain poorly understood. Here we provide evidence that different cancer-associated mutations in human DNA polymerase (Pol) ε, a major replicative DNA polymerase, impair proofreading activity to dramatically different degrees. We further provide evidence that the degree of proofreading impairment corresponds to the level of cellular mutagenesis. The main goal of this project is to test our central hypothesis that observed tumor ultramutator phenotypes result mainly from the suppression of intrinsic proofreading caused by cancer-derived mutations in Pol ε . This hypothesis is based on our preliminary data. Specifically, this project will 1) Establish a kinetic basis for cancer-causing Pol ε mutant alleles; 2) Define the mechanisms through which Pol ε exonuclease domain mutations (EDMs) generate their unique mutational signatures using novel gene-edited cell lines; and 3) Determine the mechanisms through which Pol ε variants contribute to tumor development. The proposed research is innovative due to the multidisciplinary approach that combines in vitro studies with studies using novel engineered human cell lines and mouse models to characterize the effects of cancer- associated Pol ε mutations on genome stability, mutagenesis and tumorigenesis. The novel insights into how defects at the replication fork can influence genomic alterations are also innovative. This contribution is significant because it will provide new and detailed insights into the biochemical mechanisms of how replicative DNA polymerases normally prevent the acquisition of the complex diversity of mutations found in cancer genomes, as well as provide insights into the fundamental mechanisms of DNA replication. This knowledge will deepen our understanding of cancer development and can ultimately serve to inform future studies designed to modulate DNA polymerase activities toward the goal of novel cancer therapeutic strategies.

概括癌细胞通常表现出遗传不稳定性，并积累数千至数十万其基因组中的突变。复制DNA聚合酶负责复制绝大多数核DNA。降低其准确有效合成子DNA的能力的条件分子可以直接有助于这种增加的诱变。最近，杂合的错义突变在几种肿瘤类型的DNA聚合酶（POL）ε的核酸外切酶域中鉴定出来。这些肿瘤包含迄今为止肿瘤基因组中鉴定出的最高数量的体细胞突变。尽管有这些意义后果，驱动这种诱变的机制以及最终如何影响肿瘤发生保持不当理解。在这里，我们提供的证据表明人DNA中与癌症相关的突变不同聚合酶（POL）ε是一种主要的复制性DNA聚合酶，损害了校对活性极大的不同学位。我们进一步提供了证据，表明校对程度与细胞诱变。该项目的主要目的是测试我们观察到的肿瘤超塑料的中心假设表型主要是由于抑制了由癌症衍生引起的固有校对 Polε的突变。该假设基于我们的初步数据。具体来说，该项目将1）建立引起癌症的polε突变等位基因的动力学基础； 2）定义polε的机制核酸外切酶域突变（EDMS）使用新型基因编辑产生其独特的突变特征细胞系； 3）确定polε变体有助于肿瘤发育的机制。拟议的研究具有创新性，因为多学科方法结合了体外研究通过使用新型工程人类细胞系和小鼠模型的研究来表征癌症的影响有关基因组稳定性，诱变和肿瘤发生的相关polε突变。小说对如何复制叉处的缺陷会影响基因组改变也是创新的。这个贡献是意义重大，因为它将为复制性的生化机制提供新的和详细的见解 DNA聚合酶通常会阻止癌症中发现的复杂多样性基因组，并提供有关DNA复制基本机制的见解。这些知识会加深我们对癌症发展的理解，并最终可以为未来的研究提供旨在的研究调节DNA聚合酶活性以新型癌症治疗策略的目标。