Translesional synthesis on a DNA template containing N-2-methyl-2 '-deoxyguanosine catalyzed by the Klenow fragment of Escherichia coli DNA polymerase I

Formaldehyde is produced in most living systems and is present in the envir onment. Evidence that formaldehyde causes cancer in experimental animals in fers that it may be a carcinogenic hazard to humans. Formaldehyde reacts wi th the exocyclic amino group of deoxyguanosine, resulting in the formation of N-2-methyl-2'-deoxyguanosine (N-2-Me-dG) via reduction of the Schiff bas e. The same reaction is likely to occur in living cells, because cells cont ain endogenous reductants such as ascorbic acid and gluthathione. To explor e the miscoding properties of formaldehyde-derived DNA adducts a site-speci fically modified oligodeoxynucleotide containing a N-2-Me-dG was prepared a nd used as the template in primer extension reactions catalyzed by the Klen ow fragment of Escherichia coli DNA polymerase I. The primer extension reac tion was slightly stalled one base before the N-2-Me-dG lesion, but DNA syn thesis past this lesion was readily completed. The fully extended products were analyzed to quantify the miscoding specificities of N-2-Me-dG. Prefere ntial incorporation of dCMP, the correct base, opposite the lesion was obse rved, along with small amounts of misincorporation of dTMP (9.4%). No delet ions were detected. Steady-state kinetic studies indicated that the frequen cy of nucleotide insertion for dTMP was only 1.2 times lower than for dCMP and the frequency of chain extension from the 3'-terminus of a dT:N-2-Me-dG pair was only 2.1 times lower than from a dC:N-2-Me-dG pair. We conclude t hat N-2-Me-dG is a miscoding lesion capable of generating G-->A transition mutations.