MISINCORPORATION OF NUCLEOTIDES OPPOSITE 5-MEMBERED EXOCYCLIC RING GUANINE DERIVATIVES BY ESCHERICHIA-COLI POLYMERASES IN-VITRO AND IN-VIVO- 1,N-2-ETHENOGUANINE, 5,6,7,9-TETRAHYDRO-9-OXOIMIDAZO[1,2-ALPHA]PURINE, AND ETRAHYDRO-7-HYDROXY-9-OXOIMIDAZO[1,2-ALPHA]PURINE, AND TETRAHYDRO-7-HYDROXY-9-OXOIMIDAZO[1,2-ALPHA]PURINE

A variety of exocyclic modified bases have been shown to be formed in DNA from various procarcinogens (e.g., acrolein, malonaldehyde, vinyl chloride, urethan) and are also found in untreated animals and humans, presumably arising as a result of lipid peroxidation, 1,N-2-Ethenogua nine (1,N-2-epsilon-Gua), a product known to be formed from several 2- carbon electrophiles, was placed in a known site (6256) in bacteriopha ge M13MB19 and mutations were analyzed in Escherichia coli, with 2.05% G-->A, 0.74% G-->7, and 0.09% G-->C changes found in uvrA(-) bacteria . 7,9-Tetrahydro-7-hydroxy-9-oxoimidazo[1,2-a]purine (HO-ethanoGua), f ormally the hydrated derivative of 1,N-2-epsilon-Gua, is a stable DNA product also derived from vinyl halides. When this base was placed in the same context, the mutation rate was 0.007-0.19% for G-->A, C, or T changes. The saturated etheno ring derivative of 1,N-2-epsilon-Gua, 5 ,6,7,9-tetrahydro-9-oxoimidazo[1,2-a]purine (ethanoGua) produced G-->A and G-->T mutations (0.71% each). All mutants were SOS-dependent and were attenuated by uvrA activity in E. coli. In vitro studies with fou r polymerases showed strong blocks to addition beyond the adduct site in the order ethanoGua > HO-ethanoGua > 1,N-2-epsilon-Gua. Both E. col i polymerases (pol) I exo(-) and II exo(-) and bacteriophage pol T7 ex o(-) showed extensive misincorporation opposite ethanoGua in vitro, wi th pol I exo-incorporating G and T, pol II exo(-) incorporating A, and pol T7 exo(-) incorporating A and G. All modified bases reduced the u se of the minus strand bearing the modified guanine in E. coli cells. It is of interest that even though the normal base pairing site of gua nine is completely blocked, all of the five-membered ring derivatives incorporate the normal base (C) in >80% of the replication events in E . coli. Major differences in blockage and misincorporation are seen du e to what might appear to be relatively modest structural differences, and polymerases can differ dramatically in their selectivities.