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.

抽象的 在真核生物中，DNA聚合酶（POLS）和在复制中起重要作用，但是这些pol如何 促进铅DNA链的复制尚不清楚。而广泛接受的模型 假定Pol复制领先的链，Pol复制了滞后链，我们最近发表了 Pol复制铅和滞后的DNA链的证据。然而，重要问题 与它们在复制中的作用有关的角色仍有待解决。在这里，我们提出了许多高度创新的 明确地确定Pol和Pol在复制中的作用的思想和实验方法。到 确定Pol或Pol是否复制领先的链 位于不同染色体位点的基因中的两个DNA链和多种不同的基因组的两个DNA链 酵母菌菌株，我们还将检查主要立场上是否出现了pol会产生的错误；在目标2中我们 将在POL和POL的PCNA结合结构域中使用突变来确定Pol是否在 复制这两种DNA链，或者Pol是否复制领先链，我们将进行研究 分析POL2M-644G和外切核酸酶有缺陷的POL2-4的突变器表型的遗传基础 pol突变等位基因；在AIM 3中，我们将确定是否从我们的遗传研究中指出 在重组和不匹配维修中的作用期间，将RNMP纳入领先链，而不是在 复制。总之，我们希望拟议的研究将解决与 POLS和在复制中的作用，它们将对DNA复制和相关DNA具有重要影响 修复过程以及对这些pol在基因组忠诚度中的作用的理解。