Shen J C, Rideout W M, Jones P A
Department of Biochemistry and Molecular Biology, Kenneth Norris, Jr., Comprehensive Cancer Center, University of Southern California, School of Medicine, Los Angeles 90033.
Cell. 1992 Dec 24;71(7):1073-80. doi: 10.1016/s0092-8674(05)80057-1.
HpaII methylase (M. HpaII), an example of a DNA (cytosine-5)-methyltransferase, was found to induce directly a high frequency of C-->U transition mutations in double-stranded DNA. A mutant pSV2-neo plasmid, constructed with an inactivating T-->C transition mutation creating a CCGG site, was incubated with M. HpaII in the absence of S-adenosylmethionine (SAM). This caused an approximately 10(4)-fold increase in the rate of reversion when the mutant neo plasmid was transformed into bacteria lacking uracil-DNA glycosylase. The mutation frequency was very sensitive to SAM concentration and was reduced to background when the concentration of the methyl donor exceeded 300 nM. The data support current models for the formation of a covalent complex between the methyltransferase and cytosine. They also suggest that the occurrence of mutational hot spots at CpG sites may not always be due to spontaneous deamination of 5-methylcytosine, but might also be initiated by enzymatic deamination of cytosine and proceed through a C-->U-->T pathway.
HpaII甲基化酶（M. HpaII）是一种DNA（胞嘧啶-5）-甲基转移酶，被发现可直接诱导双链DNA中高频的C→U转换突变。构建了一个突变的pSV2-neo质粒，其带有一个导致CCGG位点的失活T→C转换突变，在无S-腺苷甲硫氨酸（SAM）的情况下与M. HpaII一起孵育。当将该突变的neo质粒转化到缺乏尿嘧啶-DNA糖基化酶的细菌中时，这导致回复率增加了约10⁴倍。突变频率对SAM浓度非常敏感，当甲基供体浓度超过300 nM时，突变频率降至背景水平。这些数据支持了当前关于甲基转移酶与胞嘧啶之间形成共价复合物的模型。它们还表明，CpG位点处突变热点的出现可能并不总是由于5-甲基胞嘧啶的自发脱氨基作用，也可能由胞嘧啶的酶促脱氨基作用引发，并通过C→U→T途径进行。
