Large Loop DNA Repair in Yeast

酵母中的大环 DNA 修复

• 批准号: 6706383
• 负责人: Robert S Lahue
• 金额: $ 22.05万
• 依托单位: UNIVERSITY OF NEBRASKA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-03-01 至 2006-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6706383
• 关键词: DNA directed DNA polymerase; DNA repair; DNA replication; SDS polyacrylamide gel electrophoresis; Saccharomyces cerevisiae; antibody inhibitor; binding proteins; biotechnology; colon neoplasms; fungal genetics; fungal proteins; gender difference; genetic disorder; genetic recombination; genetic strain; hypoxanthine phosphoribosyltransferase; mass spectrometry; neoplasm /cancer genetics; nucleic acid structure; point mutation; proliferating cell nuclear antigen; protein purification; protein sequence; triple helix; western blottings

DNA mismatches are classified as either single base mispairs, like G-T, or loops of excess nucleotides on one strand. Mismatches that arise as errors in DNA replication are pre- mutagenic lesions. The well-known mismatch repair (MR) system acts on single base mispairs and on small loops to prevent substitution errors and small insertions and deletions. In the absence of MR, mutation rates are greatly elevated. The ability of MR to correct mismatches therefore provides a powerful mutation avoidance system. Humans with inherited MR deficiencies are prone to hereditary nonpolyposis colon cancer (HNPCC), as well as other types of cancer. Although MR is effective on many mispairs, it does not function on lops larger than about 13 nt. Recent biochemical evidence from yeast and human cells indicates that another repair pathway called large loop repair (LLR) is active on larger heterologies. It is proposed that LLR helps prevent large insertions and deletions by correcting the pre-mutagenic looped mispair. LLR is clearly distinct from MR; for example, LLR is fully active in cells with null mutations in crucial MR genes. The objectives of this project are to deduce key biochemical aspects of yeast LLR, to identify required proteins, and to begin establishing the importance of LLR in genetic stabilization. Two complementary approaches are proposed that utilize highly specific biochemical and genetic assays developed in this laboratory. This project will help advance the field of DNA repair by characterizing a novel, conserved, eukaryote-specific repair pathway. The research will also reveal new DNA repair proteins and provide information on their biochemical functions. This project will also address the genetic impact of LLR. Yeast was chosen because loop repair has been demonstrated in this organism, because it provides a simple eukaryotic system and because the experimental tractability of yeast allows one to answer important questions about loop repair. Based on the precedent that basic science approaches to MR in yeast led to the connection with human HNPCC, it is suggested that studies of yeast LLR may also help identify the mutational cause of other hereditary cancers or genetic diseases.

DNA错配被分类为单碱基错配，如G-T，或一条链上过量核苷酸的环。在DNA复制中出现的错配是诱变前的损伤。 众所周知的错配修复（MR）系统作用于单碱基错配和小环以防止置换错误和小插入和缺失。 在没有MR的情况下，突变率大大升高。 因此，MR纠正错配的能力提供了强大的突变避免系统。 遗传性MR缺陷的人容易患遗传性非息肉病性结肠癌（HNPCC）以及其他类型的癌症。虽然MR对许多错配有效，但它对大于约13 nt的LOPs不起作用。 最近来自酵母和人类细胞的生化证据表明，另一种称为大环修复（LLR）的修复途径在较大的异质性上是活跃的。有人提出，LLR有助于防止大的插入和缺失，通过纠正前诱变环错配。 LLR与MR明显不同;例如，LLR在关键MR基因中具有无效突变的细胞中完全活跃。 该项目的目标是推断酵母LLR的关键生化方面，以确定所需的蛋白质，并开始建立LLR在遗传稳定的重要性。提出了两种互补的方法，利用本实验室开发的高度特异性的生化和遗传检测。 该项目将通过表征一种新的、保守的、真核生物特异性的修复途径来帮助推进DNA修复领域。 这项研究还将揭示新的DNA修复蛋白，并提供有关其生化功能的信息。 该项目还将探讨LLR的遗传影响。 选择酵母是因为环修复已经在这种生物体中得到证实，因为它提供了一个简单的真核系统，因为酵母的实验易处理性允许人们回答关于环修复的重要问题。 基于基础科学方法在酵母MR导致与人类HNPCC的联系的先例，建议酵母LLR的研究也可能有助于确定其他遗传性癌症或遗传性疾病的突变原因。