O-6-ethylguanine and O-6-benzylguanine incorporated site-specifically in codon 12 of the rat H-ras gene induce semi-targeted as well as targeted mutations in Rat4 cells

To examine the miscoding properties of modified guanine residues bearing increasingly bulky O-6-substituents, Rat4 cells, grown in the presence of O-6-benzylguanine to deplete the DNA. repair protein O-6-alkylguanine-DNA alkyltransferase, were transfected with plasmids carrying H-ras genes in which O-6-methyl, O-6-ethyl- and O-6-benzylguanine were substituted for the first, second or both the first and second guanine residues of codon 12 (GGA). DNA from isolated transformed colonies was amplified by PCR and directly sequenced by high-temperature manual and automated methods, The results show that O-6-ethylguanine and O-6-benzylguanine induced semi-targeted as well as targeted mutations, in contrast to O-6-methylguanine, which induced only targeted mutations, When incorporated in place of the first guanine of H-ras codon 12, the targeted mutations induced by all these modified guanines were exclusively G-->A transitions. When incorporated at the second position of codon 12, O-6-benzylguanine induced G-->A, G-->T and G-->C mutations, O-6-Ethylguanine at the second position induced chiefly G-->A transitions, and O-6-methylguanine induced G-->A transitions exclusively, Semi-targeted mutations were strictly G-->A at the base 3' to a position 1 adduct or 5' to a position 2 adduct, The mechanism for induction of targeted mutations probably involves decreasing preference for thymidine incorporation opposite an O-6-modified guanine as the size of the O-6-substituent increases, while the mechanism for non-targeted mutations may be related to abasic site formation or to translesion synthesis which might be made error-prone by obstructive DNA lesions in this context.