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Sequence specificity of guanine alkylation and repair
M E Dolan1, M Oplinger, A E Pegg
1Department of Physiology, The Milton S. Hershey Medical Center, Pennsylvania State University, College of Medicine, Hershey 17033.
Abstract:
The sequence selectivity of methylation at the O6 and N7 position of guanine by N-methyl-N'-nitrosourea (MNU) and the rate of removal of O6-methylguanine by O6alkylguanine-DNA alkyltransferase (AGT) was determined using dodecadeoxynucleotides of defined structure. The extent of guanine adduct formed in self-complementary dodecamers, 5'-TATACGCGTATA-3', 5'-TATACCGGTATA-3' and 5'-TATAGGCCTATA-3', after methylation with [3H]MNU in a representative experiment were, respectively, 10, 19 and 30 pmol O6-methylguanine/mumol guanine and 97, 189 and 217 pmol N7-methylguanine/mumol guanine. The O6-methylguanine/N7-methylguanine ratio remained relatively constant for each dodecamer. A direct comparison between the methylation at guanine with adenine or thymine as the 5'-flanking base was made with two dodecamers, 5'-TATACATGTATA-3' and 5'-TATACTAGTATA-3'. When the guanine residue was preceded 5' by an adenine, the level of O6 and N7-alkylation was, respectively, 2.1-fold and 1.5-fold greater than when guanine was preceded 5' by a thymine. These date are consistent with a regioselective mechanism for alkylnitrosourea alkylation of guanine. The methylated dodecamer, 5'-TATACGCGTATA-3' was repaired faster than 5'-TATACCGGTATA-3' by HT29 extract containing AGT with a loss in 10 min of 0.052 pmol and 0.025 pmol O6-methylguanine, respectively. Dodecamers of the structure 5'-dCGCGAATTCm6GCG-3' and 5'-dCGCCAATTGm6GCG-3' were labeled at the 5' end with 32P by the reaction with polynucleotide kinase and after incubation with AGT, the methylated and demethylated dodecamers were separated by reversed-phase HPLC. The amount of demethylated product formed was greater for the dodecamer containing cytosine as the 5'-flanking base to O6-methylguanine compared to guanine in that same position. A higher extent of alkylation by MNU and a slower rate of repair by AGT for sites in which a guanine or modified guanine is preceded by a purine rather than a pyrimidine may explain, at least in part, mutational hot spots.