The ability of Klenow polymerase I, phage T7 polymerase (Sequenase), human polymerase alpha, and human polymerase beta to synthesize past (bypass) O6-methylguanine (O6-meG) lesions was studied in the presence of MgCl2 and MnCl2. An end-labeled 16-mer primer was annealed to the 3' end of gel-purified oligodeoxyribonucleotide templates (45-mers), each containing a single O6-meG in place of one G in the sequence -G1G2CG3G4T-. Extension products were analyzed by denaturing polyacrylamide gel electrophoresis and autoradiography. A fraction of the products extended by Klenow fragment terminated either opposite or one base before O6-meG located at sites 1 and 3. Termination occurred primarily one base before O6-meG located at sites 2 and 4. The remaining fractions that bypassed the lesions represented full-length product. In control reactions, the O6-meG-containing templates were annealed with complementary 45-mers, repaired with O6-alkylguanine DNA-alkyltransferase, annealed with an excess of labeled primer, and extended by Klenow fragment. Full-length extension of > 90% was observed with each template. Primer extension past O6-meG by DNA polymerase alpha and Sequenase was partially blocked in a manner which varied with the site of O6-meG in the template while primer extension by DNA polymerase beta was completely blocked (< 2% full length extension) with O6-meG at sites 1-4. Substitution of MnCl2 for MgCl2 in the reaction mixture greatly increased the bypass of O6-meG by Klenow fragment and DNA polymerase alpha but not Sequenase or DNA polymerase beta. The increased ability of Klenow fragment to bypass O6-meG in the presence of MnCl2 was found to result from an increased incorporation of G (O6-meG at sites 1 and 2) and A (O6-meG at sites 1, 2, and 3) opposite the lesion. The results indicate that O6-meG can block in vitro polymerization by several DNA polymerases and are consistent with the observed cytotoxic effects of methylating agents on mammalian cells.

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O6-methylguanine (O6-methylG) is believed to be the premutagenic lesion responsible for mutational activation of the H-ras proto-oncogene in rats treated with N-methyl-N-nitrosourea (MNU). Research on the repair of O6-methylG has primarily focused on the methyltransferases. Potentially, other repair proteins may be involved in repair of O6-methylG. We have investigated the effect of Escherichia coli UvrABC excision repair on O6-methylG synthesized at the rat H-ras MNU activation site in a partial rat H-ras sequence constructed in an M13mp vector. An oligonucleotide self-selection technique was used to identify progeny phage containing DNA replicated from the O6-methylG-containing strand. We found that excision repair can help protect against mutation by O6-methylG at the rat H-ras MNU activation site.

Mismatched and modified base pairs are central to questions of DNA mutation and repair. NMR and X-ray crystallography of mispairs indicate little to no local helical distortion, but these techniques are not sensitive to more global distortions of the DNA molecule. We used polyacrylamide gel electrophoresis and thermal denaturation to examine A.C, G.T, and O6-methylG.T and O6-methylG.C mismatches synthesized in place of either of two adjacent G.C base pairs in synthetic DNA duplexes. Substitution for G.C at either position decreased the stability of the duplex; O6-methylguanine was more destabilizing in place of the 5'G than in place of the 3'G. Comparisons between polymers synthesized so that lesions occurred regularly spaced on the same side of the helix and polymers synthesized so that the lesions alternated from side to side on the helix showed that these lesions introduced helical distortion composed of (i) a symmetric frictional component, probably caused by localized bubble formation, and (ii) an asymmetric component indicative of a more global effect on the DNA molecule. Comparisons between these effects at the two adjacent positions show that the extent of structural perturbation depends on sequence context.