Separation of the SOS-dependent and SOS-independent components of alkylating-agent mutagenesis.
Escherichia coli plasmids containing the rpsL+ gene (Strs phenotype) as the target for mutation were treated in vitro with N-methyl-N-nitrosourea. Following fixation of mutations in E. coli MM294A cells (recA+ Strs), an unselected population of mutant and wild-type plasmids was isolated and transferred into a second host, E. coli 6451 (recA Strr). Strains carrying plasmid-encoded forward mutations were then selected as Strr isolates, while rpsL+ plasmids conferred the dominant Strs phenotype in the second host. Mutation induction and reduced survival of N-methyl-N-nitrosourea-treated plasmids were shown to be dose dependent. Because this system permitted analysis and manipulation of the levels of certain methylated bases produced in vitro by N-methyl-N-nitrosourea, it afforded the opportunity to assess directly the relative roles of these bases and of SOS functions in mutagenesis. The methylated plasmid DNA gave a mutation frequency of 6 X 10(-5) (a 40-fold increase over background) in physiologically normal cells. When the same methylated plasmid was repaired in vitro by using purified O6-methylguanine DNA methyltransferase (to correct O6-methylguanine and O4-methylthymine), no mutations were detected above background levels. In contrast, when the methylated plasmid DNA was introduced into host cells induced by UV light for the SOS functions, rpsL mutagenesis was enhanced eightfold over the level seen without SOS induction. This enhancement of mutagenesis by SOS was unaffected by prior treatment of the DNA with O6-methylguanine DNA methyltransferase. These results demonstrate a predominant mutagenic role for alkylation lesions other than O6-methylguanine or O4-methylthymine when SOS functions are induced. The mutation spectrum of N-methyl-N-nitrosourea under conditions of induced SOS functions revealed a majority of mutagenic events at A . T base pairs.
Couto
L B
LB
Department of Chemistry, Massachusetts Institute of Technology, Cambridge 02139.
Chaudhuri
I
I
Donahue
B A
BA
Demple
B
B
Essigmann
J M
JM
D004260:DNA Repair; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics*; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial; D006147:Guanine / Q000031:analogs & derivatives / Q000032:analysis; D008770:Methylnitrosourea / Q000494:pharmacology*; D009154:Mutation; D010641:Phenotype; D010957:Plasmids; D015183:Restriction Mapping; D000091243:Ribosomal Protein S9; D013014:SOS Response, Genetics
D029968:Escherichia coli Proteins; D000091243:Ribosomal Protein S9; C000717190:RpsI protein, E coli; D006147:Guanine; D008770:Methylnitrosourea; C008449:O-(6)-methylguanine