Sequence specificity of guanine alkylation and repair

Abstract

The sequence selectivity of methylation at the O⁶ and N 7 position of guanine by N -methyl- N' -nitrosourea (MNU) and the rate of removal of O⁶ -methylguanine by O⁶ alkylguanine-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' AND 5'-TATAGGCCTATA-3', after methylation with [ ³ H]MNU in a representative experiment were, respectively, 10, 19 and 30 pmol O⁶ -methylguanine/μmol guanine and 97, 189 and 217 pmol N 7-methylguanine/μmol guanine. The O⁶ -methylguanine/ N 7-molmethylguanine 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 N 7-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 10min of 0.052 pmol and 0.025 pmol O⁶ -methylguanine, respectively. Dodecamers of the structure 5'-dCGCGAATTCm ⁶ GCG-3' and 5'-dCGCCAATTGm ⁶ GCG-3' were labeled at the 5' end with ³² P 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.