Transcription-dependent increase in multiple classes of base substitution mutations in Escherichia coli
Transcription-dependent increase in multiple classes of base substitution mutations in Escherichia coli
复制标题
DOI：
10.1128/jb.184.24.6866-6872.2002
复制
发表时间：
2002-12-01
影响因子：
3.2
通讯作者：
Bhagwat, AS
中科院分区：
文献类型：
--
作者：
Klapacz, J;Bhagwat, AS
我们以前表明，在大肠杆菌中的转录促进C（.）G-to-T（.）由于非转录链而不是转录链中胞嘧啶脱氨基作用增加而导致尿嘧啶的转变（A。Beletskii和A. S. Bhagwat，Proc. Natl. Acad. Sci. USA 93：13919-13924，1996）。为了研究C到T以外的突变，我们开发了一种新的遗传测定法，该测定法仅选择碱基置换突变，并且另外排除C（.）G到T（.）A过渡。这种新的遗传回复系统是基于终止密码子中的突变，并涉及对博莱霉素或卡那霉素的抗性的正选择。使用这种遗传系统，我们在这里表明，从一个强启动子的转录增加了非C到T以及C到T突变的水平。我们发现，高水平的转录增加了两个基因中三种不同序列背景下DNA修复熟练细胞中非C到T突变的水平，并且在这些条件下突变率高出2到4倍。这些增加不是由回复突变体的生长优势引起的，并且仅限于被诱导转录的基因。特别是，高水平的转录不会在E.杆菌对回复突变体的序列分析表明，在博来霉素抗性基因转录后，几种不同碱基替换的频率增加，G（.）C-to-T（.）颠换在转录该基因的细胞中占据主导地位。这些结果表明，高水平的转录促进了许多不同的自发碱基替换在E。杆菌
We showed previously that transcription in Escherichia coli promotes C (.) G-to-T (.) A transitions due to increased deamination of cytosines to uracils in the nontranscribed but not the transcribed strand (A. Beletskii and A. S. Bhagwat, Proc. Natl. Acad. Sci. USA 93:13919-13924, 1996). To study mutations other than that of C to T, we developed a new genetic assay that selects only base substitution mutations and additionally excludes C (.) G to T (.) A transitions. This novel genetic reversion system is based on mutations in a termination codon and involves positive selection for resistance to bleomycin or kanamycin. Using this genetic system, we show here that transcription from a strong promoter increases the level of non-C-to-T as well as C-to-T mutations. We find that high-level transcription increases the level of non-C-to-T mutations in DNA repair-proficient cells in three different sequence contexts in two genes and that the rate of mutation is higher by a factor of 2 to 4 under these conditions. These increases are not caused by a growth advantage for the revertants and are restricted to genes that are induced for transcription. In particular, high levels of transcription do not create a general mutator phenotype in E. coli. Sequence analysis of the revertants revealed that the frequency of several different base substitutions increased upon transcription of the bleomycin resistance gene and that G (.) C-to-T (.) A transversions dominated the spectrum in cells transcribing the gene. These results suggest that high levels of transcription promote many different spontaneous base substitutions in E. coli.