MUTATIONAL HOT SPOTS AND DEAMINATION OF 5 METHYLCYTOSINE

高频率的5-甲基胞嘧啶（5meC）突变为胸腺嘧啶占主导地位 基因自发突变谱。 序列变化编目 导致人类遗传性疾病，包括β-地中海贫血，血友病， 高胆固醇血症和癌症的研究表明， 大量突变是甲基化位点的C到T或G到A的变化， - CpG 早些时候，类似的高突变频率在以下位点被注意到： 甲基化E.杆菌 我们开发了一种简单但敏感的基因 一种可以直接选择胞嘧啶转化位点的试验 甲基化为胸腺嘧啶。 该测试可用于研究突变， 甲基化位点。coli和CpG位点。 使用该分析，我们有 表明细菌甲基转移酶M.EcoRII也可以将C脱氨基， U和5meC到T。 我们在这里提出的实验应该证实这一观察 并将其延伸。酶的催化参数- 介导的脱氨基反应将被确定并用于评估 酶介导和自发脱氨的相对重要性 反应. 我们将用我们的遗传系统来证明哺乳动物 甲基转移酶也可引起这种脱氨基作用。 我们还将 构建E.大肠杆菌，模拟被认为是 存在于癌症的早期阶段-高MTase水平和较低水平 甲基供体SAM 这些菌株将用于测试 假设在癌发生的早期阶段，MTase直接 导致C到T突变。 最后，我们将分离M. EcoRII的突变体， 增强导致突变的能力。 这些研究应该有助于 一个是评估胞嘧啶甲基转移酶在产生C到T中的作用， 突变和竞争假设的相对优点， 胞嘧啶甲基化位点突变的机制。

High frequencies of 5-methylcytosine (5meC) to thymine mutations dominate spectra of spontaneous mutations in genes. Cataloging of sequence change that cause human genetic diseases including beta-thalassemia, hemophilia, hypercholesterolemia and cancer have revealed that a disproportionately high number of mutations are C to T or G to A changes at sites of methylati - CpG. Earlier, similarly high mutation frequencies were noted at sites of methylation in E. coli. We have developed a simple, but sensitive genetic test that allows direct selection of the conversion of cytosines at sites of methylation to thymine. The test can be used to study mutations at methylation sites in E. coli and at CpG sites. Using this assay, we have shown that a bacterial methyltransferase, M.EcoRII, can also deaminate C to U and 5meC to T. We propose here experiments that should confirm this observation biochemically and extend it. The catalytic parameters for the enzyme- mediated deamination reactions will be determined and used to asses the relative importance of the enzyme-mediated and the spontaneous deamination reactions. We will use our genetic system to show that mammalian methyltransferases can also cause such deaminations. We will also construct strains of E. coli that mimic the conditions that are thought to exist during early stages of cancer- high MTase levels and a lower levels of the methyl donor, SAM. These strains will be used to test the hypothesis that at early stages in carcinogenesis, the MTase directly causes C to T mutations. Finally, we will isolate mutants of M.EcoRII will enhanced ability to cause mutations. Together these studies should help one evaluate the role of cytosine methyltransferases in creating C to T mutations and the relative merits of competing hypotheses regarding the mechanism of mutations at sites of cytosine methylation.