Investigation of human DNA polymerase epsilon variants

摘要 癌细胞通常表现出遗传不稳定，并积累数千至数十万 它们基因组中的突变。复制DNA聚合酶负责复制绝大多数 核DNA。降低他们准确有效地合成子DNA的能力的条件 分子可以直接促进这种增加的突变。最近，杂合性错义突变 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到。这些肿瘤 含有迄今为止在肿瘤基因组中发现的最高数量的体细胞突变。尽管有这些重要的 结果，驱动这种突变的机制，以及这最终如何影响肿瘤的形成 人们对此仍然知之甚少。在这里，我们提供了人类DNA中与癌症相关的不同突变的证据 聚合酶(POL)ε是一种主要的复制型DNA聚合酶，它会削弱校对活性，使 学位。我们进一步提供的证据表明，校对损害的程度与 细胞诱变。 这个项目的主要目标是测试我们的中心假设，即观察到肿瘤超突变者 表型主要是由癌症引起的内在校对抑制所致 POLε基因突变。这一假设是基于我们的初步数据。具体地说，这个项目将1)建立 致癌POLε突变等位基因的动力学基础；2)确定POLε 核酸外切酶结构域突变(EDM)使用新的基因编辑来生成其独特的突变特征 以及3)确定POLε变异促进肿瘤发展的机制。 由于结合了体外研究的多学科方法，建议的研究是创新的。 通过使用新的工程人类细胞系和小鼠模型来表征癌症的影响的研究- 相关的POLε突变对基因组稳定性、突变和肿瘤发生的影响。这部小说深入了解了 复制分叉上的缺陷会影响基因组的改变，这也是创新的。这一贡献是 意义重大，因为它将为复制如何复制的生化机制提供新的和详细的见解 DNA聚合酶通常阻止癌症中发现的突变的复杂多样性的获得。 基因组，以及提供对DNA复制的基本机制的洞察。这一知识将会 加深我们对癌症发展的理解，并最终有助于为未来的研究提供信息 调节DNA聚合酶活性，以达到新的癌症治疗策略的目标。

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.