Rates of individual steps in the removal of alkyl groups from O 6-methyl (Me) and -benzyl (Bz) guanine in oligonucleotides by human O6-alkylguanine DNA alkyltransferase (AGT) were estimated using rapid reaction kinetic methods. The overall reaction yields hyperbolic plots of rate versus AGT concentration for O6-MeG but linear plots for the O 6-BzG reaction, which is ∼100-fold faster. The binding of AGT and DNA (double-stranded 30-mer/36-mer complex) appears to be diffusion-limited. The rate of dissociation of the complex is ∼25-fold slower (∼1 s -1) for DNA containing O6-MeG or O6-BzG than unmodified DNA. The fluorescent dC-analog 6-methylpyrrolo[2,3-d]pyrimidine-2(3H) one deoxyribonucleoside (pyrrolo dC), which pairs with G, was positioned opposite G, O6-MeG, or O6-BzG and used as a probe of the rate of base flipping. A rapid increase of fluorescence (k ∼200 s -1) was observed with O6-HeG and O6-BzG and AGT but not with a Gly mutation at Arg128, which has been implicated in base flipping with crystal structures. Only weak and slower fluorescence changes were observed with G:pyrrolo dC or T:2-aminopurine pairs. These rate estimates were used in a kinetic model in which AGT binds and scans DNA rapidly, flips O6-alkylG residues, transfers the alkyl group in a chemical step that is rate-limiting in the case of O6-MeG but not O6-BzG, and releases the dealkylated DNA. The results explain the overall patterns of rates of alkyl group removal versus AGT concentration and the effects of the mutations, as well as the greater affinity of AGT for DNA with O 6-alkylG lesions.
All Science Journal Classification (ASJC) codes
- Molecular Biology
- Cell Biology