MUTAGENIC PATHWAYS INVOLVING 5-METHYLCYTOSINE

涉及 5-甲基胞嘧啶的诱变途径

• 批准号: 6106122
• 负责人: EDWARD L LOECHLER
• 金额: $ 14.56万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-09-01 至 1999-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6106122
• 关键词: 5 methylcytosine; DNA damage; DNA repair; adenine; alkylating agents; alkylation; amination; free radical oxygen; guanine; mutagens; point mutation; polymerase chain reaction; ribosomal RNA; thymidine

Spontaneous mutagenesis contributes significantly to genome instability in all organisms. It is essential to understand the mechanisms of spontaneous mutagenesis both for this reason and in order to be able to distinguish spontaneous mutations from those induced by exogenous agents. The goal of this project is to investigate mechanisms for GC->AT mutations observed at sites in genomes containing 5-methylcytosine (m5C), which are generally regarded to be due to "spontaneous" events. It has been known for almost 2 decades that m5C sites can be mutagenic hotspots in E.coli, and it appears that mutagenesis at m5C in human cells can be an important pathway that contributes to both tumorigenesis and germ line mutagenesis. One important component of this process appears to be m5C deamination to thymine to give a G:T mispair, which might ultimately give rise to a m5C:G->T:A mutation. In organisms which have m5C in their genomes there are (or appear to be) specific DNA repair pathways solely devoted to solving the problem of m5C deamination to T. The main question posed in this proposal is: why--in spite of a repair system specifically designed to combat this problem--does m5C:G->T:A remain an unusually prevalent pathway of mutagenesis? In this regard two lines of questions are pursued: (l) does the presence of other pathways of DNA repair that compete with the repair of m5C->T deaminations frustrate the effectiveness of the latter; and (2) are there alternatives to m5C deamination that might contribute and perhaps even dominate m5C mutagenesis in some contexts? In this regard one specific aim is directed toward evaluating whether other mismatch repair pathways might prevent effective repair of m5C deamination. A second specific aim is devoted to evaluating whether other pathways may contribute significantly to mutagenesis from m5C. For example, misreplication to give a m5C:A mispair might be unusually frequent; alternatively, this mispair might be repaired ineffectively. Finally, if any of the projects in this Program Project generates data to suggest that a particular lesion is important to spontaneous mutagenesis, a site-specific study of that lesion will be conducted. Most of the studies proposed herein will be done in E. coli. The studies are only feasible given the development of the enabling technology, CDCE/hifi PCR, which significantly reduces the time and effort required to determine a mutational spectrum.

自发突变显着导致基因组不稳定 所有生物体。了解自发性机制非常重要 诱变既是出于这个原因，也是为了能够区分 由外源物质诱导的自发突变。目标是 该项目旨在研究 GC->AT 突变的机制 基因组中含有 5-甲基胞嘧啶 (m5C) 的位点，通常是 被认为是“自发”事件造成的。它几乎已经被人们所熟知 20 年来，m5C 位点可能是大肠杆菌中的诱变热点，并且它 看来人类细胞中 m5C 的突变可能是一个重要途径 这有助于肿瘤发生和种系突变。一 该过程的重要组成部分似乎是 m5C 脱氨 胸腺嘧啶产生 G:T 错配，这可能最终导致 m5C:G->T:A 突变。在基因组中有 m5C 的生物体中 是（或似乎是）特定的 DNA 修复途径，专门用于 解决m5C脱氨到T的问题。提出的主要问题 这个建议是：为什么——尽管有专门设计的修复系统 为了解决这个问题——m5C:G->T:A 仍然是异常流行的吗 诱变途径？在这方面，提出了两方面的问题： (l)是否存在与DNA修复竞争的其他途径 m5C->T 脱氨基的修复破坏了 后者； (2) 是否有 m5C 脱氨基的替代方案 在某些情况下促进甚至主导 m5C 突变？在 在这方面，一个具体目标是评估其他方面是否 错配修复途径可能会阻碍 m5C 的有效修复 脱氨作用。第二个具体目标致力于评估其他 途径可能对 m5C 的诱变有显着贡献。为了 例如，错误复制给出 m5C：错配可能是不常见的 经常；否则，这种错误可能无法有效修复。 最后，如果该计划项目中的任何项目生成数据 表明特定的损伤对于自发突变很重要， 将对该病变进行特定地点的研究。大部分的 本文提出的研究将在大肠杆菌中进行。研究仅 考虑到使能技术 CDCE/hifi PCR 的发展，这是可行的， 这大大减少了确定所需的时间和精力 突变谱。