MUTAGENIC PATHWAYS INVOLVING 5-METHYLCYTOSINE

自发突变极大地促进了人类基因组的不稳定。 所有的有机体。了解自发性的机制是很重要的。 突变既是出于这个原因，也是为了能够区分 由外源诱变剂诱发的自发突变。的目标是 该项目旨在研究观察到的GC-&GT；AT突变的机制 基因组中含有5-甲基胞嘧啶(M5C)的位置，通常是 被认为是由“自发”事件引起的。它几乎已经为人们所熟知 20年来，M5C位点可以成为大肠杆菌中的突变热点，而它 人类细胞中M5C的突变可能是一条重要的途径 这既有助于肿瘤的发生，也有助于生殖系的突变。一 这一过程的重要组成部分似乎是M5C脱氨基 胸腺嘧啶产生G：T错配，这可能最终导致 M5C：G-&GT；T：一种突变。在基因组中含有M5C的生物体中 是否(或似乎是)特定的DNA修复途径专门用于 解决了M5C脱氨为T的问题。 这项建议是：为什么--尽管有专门设计的维修系统 为了解决这个问题--M5C：G-&T；A是否仍然异常普遍 诱变途径？在这方面，提出了两个问题： (L)是否存在其他与之竞争的DNA修复途径 M5C-和GT；T的修复会挫败 后者；以及(2)有没有替代M5C脱氨的方法 在某些情况下促成甚至主导了M5C突变？在……里面 在这方面，一个具体的目标是评估其他 错配修复途径可能会阻碍M5C的有效修复 去氨基。第二个具体目标是评估其他人是否 途径可能对M5C的突变有重要作用。为 例如，错误复制以获得M5C：错误配对可能是不寻常的 频繁；或者，这种错配可能被无效地修复。 最后，如果此计划项目中的任何项目生成数据以 表明特定的损伤对自发突变很重要， 将对该病变进行特定部位的研究。大多数 本文建议的研究将在大肠杆菌中进行。这些研究只是 鉴于使能技术CDCE/HIFI聚合酶链式反应的发展是可行的， 这大大减少了确定 突变谱。

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.