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: 9757794
• 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/9757794
• 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(POL)和在复制中起着重要作用，但这些POL是如何 对主要DNA链的复制起作用的因素尚不清楚。虽然被广泛接受的模型 假设POL复制前导链，POL复制滞后链，我们最近发表了这篇文章 有证据表明POL同时复制领先和落后的DNA链。然而，重要的问题是 关于它们在复制中的作用的问题仍有待解决。在这里，我们提出了一些极具创新性的 明确确定POL和POL在复制中的作用的想法和实验方法。至 确定POL或POL是否复制了前导链，在目标1中，我们将分析POL生成的错误 这两条DNA链在基因中位于不同的染色体位置，并在全基因组范围内存在一些不同 酵母菌株，我们还将检查POL产生的错误是否发生在领先的支架上；在目标2中，我们 将使用POL和POL的增殖细胞核抗原结合域的突变来确定POL是否在 复制两条dna链或是否复制Pol的前导链，我们将进行研究以 分析pol2M-644G突变表型和外切酶缺陷pol2-4的遗传基础 POL突变等位基因；在目标3中，我们将确定根据我们的遗传研究，POL是否 在重组和错配修复过程中，而不是在过程中，将rNMP结合在前导链上 复制。综上所述，我们预期拟议的研究将会解决与 POLS和在复制中的作用，它们将对DNA复制和相关的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。