Abstract
Rates of individual steps in the removal of alkyl groups from O6-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 O6-BzG reaction, which is approximately 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 approximately 25-fold slower (approximately 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 approximately 200 s(-1)) was observed with O6-MeG 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 O6-alkylG lesions.
Highlights
DNA is subject to damage by endogenous and exogenous chemicals, which generate DNA adducts or otherwise altered DNA bases
These results imply that the size of the alkylguanine-DNA transferase (AGT) active site is crucial for substrate recognition
We studied the rates of alkyl transfer, dissociation, and base flipping for O6-MeG and O6-BzG adducts in a double-stranded oligonucleotide with AGT
Summary
Chemicals—UDG, piperidine, and O6-BzG were obtained from Sigma-Aldrich; [␥-32P]ATP was purchased from PerkinElmer Life Sciences. Sample syringe A (12.5 l) included a preincubated solution of AGT protein in Tris-HCl buffer (50 mM, pH 7.8) containing 1 mM DTT, 100 g of bovine serum albumin mlϪ1, and 32P-labeled target DNA; either 0.05 M O6-BzG DNA, 0.2 M O6-MeG DNA, or 1.0 M unmodified DNA, with AGT to DNA ratios indicated in the tables. After the samples were mixed and incubated for times varying from 0.05 to 30 s at 25 °C, polymerization was initiated by the addition of dNTP (200 M) and MgCl2 (5 mM) from the central drive syringe, with a constant reaction time of 0.25 s. Samples were analyzed by gel electrophoresis (16% polyacrylamide (w/v), 80 watts, 3.0 h) and radioactivity in the resolved bands was quantitated using a Molecular Imager FX phosphorimaging system. An excitation wavelength of 360 nm was used in assays measuring pyrrolo dC fluorescence.
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