Misincorporation of nucleotides opposite five-membered exocyclic ring guanine derivatives by escherichia coli polymerases in vitro and in vivo: 1,N2-ethenoguanine, 5,6,7,9-tetrahydro-9-oxoimidazo[1, 2-a]purine, and 5,6,7,9-tetrahydro-7-hydroxy-9-oxoimidazo[1, 2-a]purine - PubMed (original) (raw)

. 1998 Apr 14;37(15):5184-93.

doi: 10.1021/bi972327r.

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Misincorporation of nucleotides opposite five-membered exocyclic ring guanine derivatives by escherichia coli polymerases in vitro and in vivo: 1,N2-ethenoguanine, 5,6,7,9-tetrahydro-9-oxoimidazo[1, 2-a]purine, and 5,6,7,9-tetrahydro-7-hydroxy-9-oxoimidazo[1, 2-a]purine

S Langouët et al. Biochemistry. 1998.

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Abstract

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, N2-Ethenoguanine (1,N2-epsilon-Gua), a product known to be formed from several 2-carbon electrophiles, was placed in a known site (6256) in bacteriophage M13MB19 and mutations were analyzed in Escherichia coli, with 2.05% G-->A, 0.74% G-->T, and 0.09% G-->C changes found in uvrA- bacteria. 5,6,7, 9-Tetrahydro-7-hydroxy-9-oxoimidazo[1,2-a]purine (HO-ethanoGua), formally the hydrated derivative of 1,N2-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, N2-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 four polymerases showed strong blocks to addition beyond the adduct site in the order ethanoGua > HO-ethanoGua > 1,N2-epsilon-Gua. Both E. coli polymerases (pol) I exo- and II exo- and bacteriophage pol T7 exo- showed extensive misincorporation opposite ethanoGua in vitro, with 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 use 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 guanine 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 due to what might appear to be relatively modest structural differences, and polymerases can differ dramatically in their selectivities.

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