ROLES OF TRANSCRIPTION AND REPAIR IN ALKYLATION MUTAGENESIS
ROLES OF TRANSCRIPTION AND REPAIR IN ALKYLATION MUTAGENESIS
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DOI：
10.1016/0921-8777(94)90071-x
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发表时间：
1994-05-01
期刊：
影响因子：
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通讯作者：
SEKIGUCHI, M
中科院分区：
文献类型：
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作者：
ITO, T;NAKAMURA, T;SEKIGUCHI, M
利用克隆在高拷贝数质粒上的大肠杆菌rpsL基因正向突变的分析方法，分析了暴露于烷化剂的大肠杆菌细胞中发生的突变。从野生型和O-6-甲基鸟嘌呤甲基转移酶缺陷突变(ADA(-)OGT(-))细胞中恢复了N-甲基-N‘-硝基-N-亚硝基(MNNG)诱导的突变，并测定了它们的序列变化。我们发现，从野生型菌株恢复的突变主要是位于rpsL序列中几个热点的G：C到A：T转变。绝大多数突变是在靶基因转录链上的鸟嘌呤残基和胸腺嘧啶残基上发现的。尽管甲基转移酶突变体在诱变效应和细胞杀伤效应方面对烷基化试剂表现出超敏反应，但从MNNG处理的ADA(-)OGT(-)细胞中恢复的rpsL(-)突变的类别和位置分布与MNNG处理的野生型细胞相似。因此，MNNG诱导的rpsL(-)突变的位点选择性似乎不是由于甲基转移修复酶的特异性，而可能是由于突变损伤(O-6-甲基鸟嘌呤)在靶序列中的分布。SN2烷基化试剂甲烷磺酸甲酯诱发的突变在rpsL系统中显示出相似的类别和位置分布。当rpsL基因的转录水平比真正的rpsL启动子启动的转录水平低120倍时，MNNG诱导的突变的位点选择性显著改变。在此条件下，78%的突变发生在5‘-GG(A或C)-3’的中心鸟嘌呤处，其中2/3位于rpsL基因的非转录链上。这些结果表明，MNNG诱导的突变的位置选择性至少由三个因素决定：(I)对鸟嘌呤残基的化学反应的侧翼碱基效应，(Ii)转录的链特异性修复，可能是通过UvrABC系统，以及(Iii)目标基因的转录对DNA烷基化和链特异性修复的影响。
Mutations occurring in Escherichia coli cells exposed to alkylating agents have been analyzed using an assay for forward mutations in the E. coli rpsL gene cloned on a high copy number plasmid. N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced mutations were recovered from wild-type and O-6-methylguanine methyltransferase-deficient mutant (ada(-) ogt(-)) cells and their sequence alterations determined. We found that the mutations recovered from the wild-type strain were predominantly G:C to A:T transitions located at several hot spots in the rpsL sequence. A vast majority of the mutations were found at guanine residues preceded by thymine on the transcribed strand of the target gene. Although the methyltransferase mutant showed hypersensitivity to the alkylating reagent in terms of mutagenic effect and cell killing effects, the class and site distributions of the rpsL(-) mutations recovered from MNNG-treated ada(-) ogt(-) cells were similar to those observed with MNNG-treated wild-type cells. Therefore, the site preference of MNNG-induced rpsL(-) mutations seems to be due not to the specificity of methyl-transferring repair enzymes but probably to the distribution of the mutagenic lesions (O-6-methylguanine) in the target sequence. Mutations induced by methyl methanesulfonate, an SN2 alkylating agent, showed similar class and site distributions in the rpsL system. The site preference of MNNG-induced mutations was significantly changed when the level of transcription of the rpsL gene was decreased to 120-fold lower than that promoted by the authentic rpsL promoter. Under these conditions, 78% of mutations were induced at the central guanine of 5'-GG(A or C)-3' and 2/3 of them were on the non-transcribed strand of the rpsL gene. These results suggested that the site preference of MNNG-induced mutations is determined by at least three factors: (i) a flanking-base effect on the chemical reactivity of a guanine residue, (ii) transcribed strand-specific repair, probably by the UvrABC system, and (iii) the effects of transcription of the target gene on the alkylation of DNA and the strand-specific repair.