Investigation of human DNA polymerase epsilon variants

人类 DNA 聚合酶 epsilon 变异体的研究

• 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
• 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

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.

总结 癌细胞通常表现出遗传不稳定性，并积累数千至数十万个癌细胞。 基因组中的突变复制型DNA聚合酶负责复制绝大多数的 核DNA降低其准确和有效合成子DNA的能力的条件 分子可以直接促成这种增加的诱变。最近，杂合错义突变 在几种肿瘤类型的DNA聚合酶（Pol）ε的核酸外切酶结构域中鉴定出了这些基因。这些肿瘤 包含迄今为止在肿瘤基因组中鉴定的最高数量的体细胞突变。尽管取得了这些重大 结果，驱动这种诱变的机制以及这最终如何影响肿瘤发生 仍然知之甚少。在这里，我们提供的证据表明，人类DNA中不同的癌症相关突变 聚合酶（Pol）ε，一种主要的复制DNA聚合酶，损害校对活性，以显著不同 度我们进一步提供证据表明，校对减值的程度对应的水平， 细胞诱变 这个项目的主要目标是检验我们的中心假设，即观察到的肿瘤超变因子 表型的结果主要是由于抑制的内在校对所造成的癌症衍生的 Pol ε突变这个假设是基于我们的初步数据。具体而言，该项目将1）建立 导致癌症的Pol ε突变等位基因的动力学基础; 2）定义Pol ε突变的机制。 核酸外切酶结构域突变（EDM）使用新的基因编辑技术产生其独特的突变特征。 细胞系;和3）确定Pol ε变体促进肿瘤发展的机制。 拟议的研究是创新的，由于多学科的方法，结合在体外研究 使用新的工程人类细胞系和小鼠模型来表征癌症的影响， 相关Pol ε突变对基因组稳定性、诱变和肿瘤发生的影响。这部小说深入探讨了 复制叉上的缺陷会影响基因组的改变也是创新的。这种贡献 意义重大，因为它将提供新的和详细的见解如何复制的生化机制， DNA聚合酶通常阻止癌症中发现的复杂多样性突变的获得 基因组，以及提供深入了解DNA复制的基本机制。这些知识将 加深我们对癌症发展的理解，并最终为未来的研究提供信息， 调节DNA聚合酶活性，以实现新的癌症治疗策略的目标。