Wyszynski M, Gabbara S, Bhagwat A S
Department of Chemistry, Wayne State University, Detroit, MI 48202.
Proc Natl Acad Sci U S A. 1994 Feb 15;91(4):1574-8. doi: 10.1073/pnas.91.4.1574.
Sites of cytosine methylation are hot spots for C to T mutations in Escherichia coli DNA. We have developed a genetic reversion assay that allows direct selection of C to T mutations at a site of methylation. Because the mutant gene is on a plasmid, this system can be used to study mutational effects of biochemical agents in vitro as well as in vivo. Using this system we show that in vitro an E. coli methyltransferase can cause C to U deaminations at a site of methylation. Reaction conditions that are known to inhibit a side reaction of the methyltransferase also suppress reversion frequency, suggesting that this side reaction is required for deamination. Furthermore, a mutation in the enzyme that eliminates its catalytic activity but not its ability to bind DNA eliminates the ability of the enzyme to cause C to U deaminations. Despite this, in vivo experiments strongly suggest that enzyme-catalyzed deaminations of cytosine do not play a major role in making methylation sites in E. coli hot spots for mutations. For example, although uracil-DNA glycosylase (Ung) suppresses the occurrence of mutations due to C to U deaminations, the frequency of C to T mutations at a methylation site remains high in ung+ cells. Furthermore, the reversion frequencies in ung+ and ung- cells are quite similar.
胞嘧啶甲基化位点是大肠杆菌DNA中C到T突变的热点。我们开发了一种遗传回复突变检测方法，可直接选择甲基化位点处的C到T突变。由于突变基因位于质粒上，该系统可用于研究生化试剂在体外和体内的突变效应。利用该系统我们发现，在体外，大肠杆菌甲基转移酶可在甲基化位点导致C到U的脱氨基作用。已知抑制甲基转移酶副反应的反应条件也会抑制回复突变频率，这表明该副反应是脱氨基作用所必需的。此外，酶中的一个突变消除了其催化活性但未消除其结合DNA的能力，该突变也消除了酶导致C到U脱氨基的能力。尽管如此，体内实验强烈表明，胞嘧啶的酶催化脱氨基作用在使大肠杆菌中的甲基化位点成为突变热点方面并不起主要作用。例如，尽管尿嘧啶-DNA糖基化酶（Ung）可抑制由于C到U脱氨基导致的突变发生，但在ung+细胞中，甲基化位点处C到T突变的频率仍然很高。此外，ung+和ung-细胞中的回复突变频率非常相似。
