Kinetics of O⁶-Pyridyloxobutyl-2′-deoxyguanosine Repair by Human O⁶-alkylguanine DNA Alkyltransferase

Abstract

Tobacco-specific nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N-nitrosonicotine (NNN) are potent carcinogens believed to contribute to the development of lung tumors in smokers. NNK and NNN are metabolized to DNA-reactive species that form a range of nucleobase adducts, including bulky O6-[4-oxo-4-(3-pyridyl)but-1-yl]deoxyguanosine (O6-POB-dG) lesions. If not repaired, O6-POB-dG adducts induce large numbers of G → A and G → T mutations. Previous studies have shown that O6-POB-dG can be directly repaired by O6-alkylguanine-DNA alkyltransferase (AGT), which transfers the pyridyloxobutyl group from O6-alkylguanines in DNA to an active site cysteine residue within the protein. In the present study, we investigated the influence of DNA sequence context and endogenous cytosine methylation on the kinetics of AGT-dependent repair of O6-POB-dG in duplex DNA. Synthetic oligodeoxynucleotide duplexes containing site-specific O6-POB-dG adducts within K-ras and p53 gene-derived DNA sequences were incubated with recombinant human AGT protein, and the kinetics of POB group transfer was monitored by isotope dilution HPLC-ESI+-MS/MS analysis of O6-POB-dG remaining in DNA over time. We found that the second order rates of AGT-mediated repair were influenced by DNA sequence context (10-fold differences), but were only weakly affected by the methylation status of neighboring cytosines. Overall, AGT-mediated repair of O6-POB-dG was 2–7 times slower than that of O6-Me-dG adducts. To evaluate the contribution of AGT to O6-POB-dG repair in human lung, normal human bronchial epithelial cells (HBEC) were treated with model pyridyloxobutylating agent, and O6-POB-dG adduct repair over time was monitored by HPLC-ESI+-MS/MS. We found that HBEC cells were capable of removing O6-POB-dG lesions, and the repair rates were significantly reduced in the presence of AGT inhibitor (O6-benzylguanine). Taken together, our results suggest that AGT plays an important role in protecting human lung against tobacco nitrosamine-mediated DNA damage and that inefficient AGT repair of O6-POB-dG at specific sequence contributes to mutational spectra observed in smoking-induced lung cancer.

6‐phenylpyrrolocytosine as a fluorescent probe to examine nucleotide flipping catalyzed by a DNA repair protein

“…The hyperbolic fit to the 1/τ 1 values shown in Figure 3 must be considered to be approximate because the experimental conditions limited the range of AGT concentrations that could be employed while maintaining pseudo‐first order conditions. However, a K d for AGT binding of 560 ± 40 nM (5 °C) is in reasonable agreement with the value reported previously (1.5 μM, 22 °C), [ 39 ] which was determined using a different technique (gel shift assay) that includes the energetic contributions from all steps in the binding and flipping processes. The forward rate constant for the second step of k 2 = 530 ± 40 seconds −1 at 5 °C (equivalent to a second order constant of ~3 x 10 8 M −1 second −1 for an AGT concentration of 1.6 μM) is more than two orders of magnitude greater than the repair rate constant for AGT with this modified DNA (1.79 x 10 6 M −1 second −1 ).…”

“…The forward rate constant for the second step of k 2 = 530 ± 40 seconds −1 at 5 °C (equivalent to a second order constant of ~3 x 10 8 M −1 second −1 for an AGT concentration of 1.6 μM) is more than two orders of magnitude greater than the repair rate constant for AGT with this modified DNA (1.79 x 10 6 M −1 second −1 ). [ 39,40 ] The rate constants for binding ( k 1 ) and release ( k −1 ) of AGT must exceed this value in order to yield the observed hyperbolic dependence. These rate constants presumably report on both AGT binding and one‐dimensional sliding on the DNA to find the alkylated base.…”

“…However, one or probably both exceed the previously reported pseudo‐first order rate constant for the overall reaction employing 1.6 μM AGT (1.6 x 10 −6 M −1 x 1.79 x 10 6 M −1 second −1 = ~2.9 seconds −1 , 22 °C), and thus they are kinetically competent steps in the process. [ 39 ]…”

“…[ 9 ] Using a mass spectrometry‐based assay, we had observed a decrease in the rates of AGT repair of O 6 ‐alkyl‐G containing DNA when Me C was introduced immediately 5′ to the adduct. [ 9,39 ] However, our previous stopped‐flow fluorescence experiments to study nucleotide flipping showed that the rate of nucleotide flipping was not significantly affected by this cytosine methylation. [ 9 ] This discrepancy may stem from the use of pyrrolo‐C in the earlier study, which is less sensitive than 6‐phenylpyrrolo‐C to its local environment.…”

Impact of tobacco-specific nitrosamine–derived DNA adducts on the efficiency and fidelity of DNA replication in human cells

“…The high mutation frequency observed for the lesion suggests that it is not efficiently repaired prior to being encountered by the DNA replication machinery. Along this line, several recent studies have shown that O 6 -POB-dG can be directly repaired by O 6 -alkylguanine-DNA alkyltransferase (AGT) (33,34). Following the transfer of a methyl or other alkyl groups from O 6 -alkylguanine to an internal cysteine residue (Cys-145) of AGT, O 6 -dG adducts are repaired, and the protein becomes inactivated during the repair process (35,36).…”