MUTAGENIC PATHWAYS INVOLVING 5-METHYLCYTOSINE

自发突变显著地导致了基因组的不稳定性， 所有的有机体。理解自发性的机制是至关重要的。 为了这个原因和为了能够区分 自发突变与外源因子诱导的突变。的目标 该项目旨在研究在2010年观察到的GC->AT突变的机制。 基因组中含有5-甲基胞嘧啶（m5 C）的位点，通常是 被认为是由于“自发”事件。它已经被称为几乎 二十年来，m5 C位点可以成为大肠杆菌的致突变热点，并且它 人类细胞中m5 C的突变似乎是一个重要的途径， 导致肿瘤发生和生殖细胞突变。一 该方法的重要组成部分似乎是m5 C脱氨基， 胸腺嘧啶产生G：T错配，这可能最终导致 m5 C：G->T：A突变。在基因组中含有m5 C的生物体中， 是（或似乎是）特定的DNA修复途径， 解决了m5 C脱氨成T.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，m5 C：G->T：A是否仍然是一个异常普遍的问题？ 诱变途径？在这方面，提出了两类问题： (l)是否存在其他的DNA修复途径， m5 C->T脱氨的修复阻碍了 后者;和（2）是否有替代m5 C脱氨基，可能 在某些情况下，甚至可能主导m5 C诱变？在 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止m5 C的有效修复， 脱氨作用。第二个具体目标是评估其他 途径可能显著地促进来自m5 C的诱变。为 例如，错误复制产生m5 C：错误配对可能是不寻常的， 频繁;或者，这种错配可能无法有效修复。 最后，如果此计划项目中的任何项目生成数据， 表明特定损伤对于自发突变是重要的， 将对该病变进行特定部位研究。大部分 本文提出的研究将在E.杆菌这些研究只是 可行的，鉴于使能技术的发展，CDCE/hifi PCR， 这显著地减少了确定一个 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.