Roles of DNA polymerases delta and epsilon in replication, repair, and genomic fidelity

DNA 聚合酶 delta 和 epsilon 在复制、修复和基因组保真度中的作用

• 批准号: 9980963
• 负责人: SATYA PRAKASH
• 金额: $ 31.6万
• 依托单位: UNIVERSITY OF TEXAS MED BR GALVESTON
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-08-07 至 2022-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9980963
• 关键词: Alleles; Binding; Cells; DNA; DNA Polymerase III; DNA Repair; DNA biosynthesis; DNA-Directed DNA Polymerase; Eukaryota; Exonuclease; Gel; Generations; Genes; Genetic; Genetic study; Genomics; Hot Spot; Location; Mediating; Mismatch Repair; Modeling; Mutation; Phenotype; Play; Polymerase; Process; Publications; Publishing; Ribonucleases; Ribonucleotides; Role; Site; Surgical incisions; Techniques; Testing; Type I DNA Topoisomerases; Yeasts; alkalinity; base; genetic approach; genome-wide; innovation; mutant; next generation sequencing; novel; novel strategies; recombinational repair; repaired; replicase

Abstract In eukaryotes, DNA polymerases (Pols)  and  play important roles in replication, but how these Pols contribute to the replication of the leading DNA strand has remained unclear. While the widely accepted model posits that Pol replicates the leading strand and Pol replicates the lagging strand, we recently published evidence that Pol replicates both the leading and lagging DNA strands. Nevertheless, important issues pertaining to their roles in replication remain to be resolved. Here we propose a number of highly innovative ideas and experimental approaches to unambiguously establish the roles of Pol and Pol in replication. To determine whether Pol or Pol replicates the leading strand, in Aim 1 we will analyze Pol-generated errors on the two DNA strands in genes located at different chromosomal sites and genome-wide in a number of different yeast strains, and we will also examine whether Pol-generated errors occur on the leading stand; in Aim 2 we will use mutations in the PCNA binding domain of Pol and Pol to determine whether Pol plays a major role in replicating both DNA strands or whether Pol replicates the leading strand, and we will carry out studies to analyze the genetic basis of the mutator phenotype of the pol2M-644G and the exonuclease defective pol2-4 Pol mutant alleles; and in Aim 3, we will determine whether as indicated from our genetic studies, Pol incorporates rNMPs on the leading strand during its roles in recombination and mismatch repair, and not during replication. Altogether, we expect that the proposed studies will resolve the outstanding issues relating to the role of Pols  and  in replication, and they will have important bearing on DNA replication and associated DNA repair processes and on the understanding of the roles of these Pols in genomic fidelity.

摘要 在真核生物中，DNA聚合酶（Pols）在复制过程中起着重要作用，但这些Pols是如何在复制过程中发挥作用的呢？ 对DNA前导链复制的贡献仍然不清楚。虽然被广泛接受的模型 我们最近发表了一项研究， 证明了Pollymphocyte复制了前导和滞后DNA链。然而，重要问题 有关其在复制中的作用的问题仍有待解决。在这里，我们提出了一些高度创新的 想法和实验方法，明确建立的作用，Poll和Poll的复制。到 为了确定Pol C3或Pol C4是否复制前导链，在目标1中，我们将分析Pol C3产生的错误， 基因中的两条DNA链位于不同的染色体位点，在许多不同的基因组中， 酵母菌株，我们还将检查是否Pol产生的错误发生在领先的立场;在目标2，我们 将使用Pol β和Pol β的PCNA结合结构域中的突变来确定Pol β是否在增殖中起主要作用。 复制两条DNA链，或者是否Pol复制了前导链，我们将进行研究， 分析pol 2 M-644 G和核酸外切酶缺陷型pol 2 -4的突变体表型的遗传基础 在目标3中，我们将确定是否如我们的遗传研究所示， 在重组和错配修复过程中，在前导链上掺入rNMPs，而在 复制的总的来说，我们预期拟议的研究将解决有关 Pols β和Pols β在复制中的作用，它们将对DNA复制和相关DNA产生重要影响 修复过程和对这些Pol在基因组保真度中的作用的理解。