MUTAGENESIS IN ESCHERICHIA-COLI BY 3 O-6-SUBSTITUTED GUANINES IN DOUBLE-STRANDED OR GAPPED PLASMIDS

Plasmids were constructed with guanine (G) or O-6-methyl-(m(6)G). O-6- ethyl-(e(6)G). or O-6-benzyl-(b(6)G) guanine in the initiation codon ( ATG) of the lacZ' gene. Four deoxyuridine residues were incorporated n ear the modified guanine in the complementary strand. The deoxyuridine -containing plasmids exhibited similarly high transformation efficienc ies in ung(-) Escherichia coli, although the frequency of mutations in duced by m(6)G, e(6)G, and b(6)G residues was relatively low. Treatmen t of the plasmids with uracil-DNA glycosylase (UDG), to remove the ura cil residues, or UDG and exonuclease III, to create a gap in the deoxy uridine-containing strand, reduced transformation efficiency for adduc t-containing plasmids but did not affect transformation efficiency for control plasmids. However, the same treatments dramatically enhanced mutagenesis by m(6)G, e(6)G, and b(6)G. These results were consistent with blockage of replication by the modified guanines in double-strand ed plasmids resulting in preferential replication of the complementary strand. Replication past the modified guanines was forced in the gapp ed plasmids. The frequency of modified guanine-induced mutations in ga pped vectors was similar in strains of E, toil that were proficient in DNA polymerase III but deficient in either DNA polymerase I or II or both polymerase I and II suggesting either that, polymerase III was pr imarily responsible for adduct bypass in all strains or that the proba bility of base misinsertion during bypass by either polymerase I or II was similar to that for polymerase III, Repair studies with gapped pl asmids indicated that m(6)G was subject to repair by Ada methyltransfe rase and to posteplication processing by methylation-directed mismatch repair. Neither e(6)G nor b(6)G were similarly repaired. Both m(6)G a nd e(6)G efficiently coded for thymine incorporation although b(6)G ap peared to be less miscoding.