Comparative mutagenesis of O6-methylguanine and O4-methylthymine in Escherichia coli.
The qualitative and quantitative features of mutagenesis by two DNA adducts of carcinogenic alkylating agents, O6-methylguanine (m6G) and O4-methylthymine (m4T), were examined in vivo. The deoxyhexanucleotides 5'-GCTAGC-3' and 5'-GCTAGC-3' were synthesized, where the underlined bases are the positions of m4T or m6G, respectively. By use of recombinant DNA techniques, the respective hexanucleotides or an unmodified control were inserted into a six-base gap in the otherwise duplex genome of the Escherichia coli virus M13mp19-NheI. The duplex adducted genome was converted to single-stranded form and introduced into an E. coli strain that was phenotypically normal with regard to m6G/m4T repair, a strain deficient in repair by virtue of an insertion in the gene encoding the Ada-m6G/m4T DNA methyltransferase, or the same two cell lines after challenge with N-methyl-N'-nitro-N-nitrosoguanidine. Treatment with this alkylating agent chemically compromises alkyl-DNA repair functions. The mutation efficiency of m6G was low or undetectable (0-1.7%) in all cell systems tested, owing, we believe, to rapid repair. In striking contrast, the mutagenicity of m4T was high (12%) in cells fully competent to repair alkylation damage and was roughly doubled when those cells were pretreated with N-methyl-N'-nitro-N-nitrosoguanidine to suppress repair. Taken together, these data suggest that m4T is potentially more mutagenic than m6G and, if formed by a DNA methylating agent, may pose a significant threat to the genetic integrity of an organism.
Dosanjh
M K
MK
Department of Chemistry, Whitaker College of Health Sciences and Technology, Cambridge, Massachusetts.
Singer
B
B
Essigmann
J M
JM
D001426:Bacterial Proteins / Q000235:genetics; D001483:Base Sequence / Q000187:drug effects; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics*; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial / Q000187:drug effects; D006147:Guanine / Q000031:analogs & derivatives*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019853:O(6)-Methylguanine-DNA Methyltransferase; D013936:Thymidine / Q000031:analogs & derivatives*; D014157:Transcription Factors
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D014157:Transcription Factors; C008505:O-methylthymidine; D006147:Guanine; C008449:O-(6)-methylguanine; C045675:Ada protein, E coli; D019853:O(6)-Methylguanine-DNA Methyltransferase; D013936:Thymidine