O-6-ETHYLGUANINE AND O-6-BENZYLGUANINE INCORPORATED SITE-SPECIFICALLYIN 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 beari ng increasingly bulky O-6-substituents, Rat4 cells, grown in the prese nce of O-6-benzylguanine to deplete the DNA. repair protein O-6-alkylg uanine-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 wa s amplified by PCR and directly sequenced by high-temperature manual a nd automated methods, The results show that O-6-ethylguanine and O-6-b enzylguanine induced semi-targeted as well as targeted mutations, in c ontrast to O-6-methylguanine, which induced only targeted mutations, W hen incorporated in place of the first guanine of H-ras codon 12, the targeted mutations induced by all these modified guanines were exclusi vely G-->A transitions. When incorporated at the second position of co don 12, O-6-benzylguanine induced G-->A, G-->T and G-->C mutations, O- 6-Ethylguanine at the second position induced chiefly G-->A transition s, and O-6-methylguanine induced G-->A transitions exclusively, Semi-t argeted mutations were strictly G-->A at the base 3' to a position 1 a dduct or 5' to a position 2 adduct, The mechanism for induction of tar geted 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 wh ich might be made error-prone by obstructive DNA lesions in this conte xt.