Effect of the O6 Substituent on Misincorporation Kinetics Catalyzed by DNA Polymerases at O⁶-Methylguanine and O⁶-Benzylguanine

Abstract

Misincorporation at a DNA-carcinogen adduct may contribute to formation of mutations if a polymerase proceeds past the lesion, compromising fidelity, as in the G:C to A:T mutations caused by O(6)-alkylguanine. Replication of primer/templates containing guanine (G), O(6)-methylguanine (O(6)-MeG), or O(6)-benzylguanine (O(6)-BzG) was assessed using T7 DNA polymerase exo(-) (T7(-)) and HIV-1 reverse transcriptase (RT). The steady-state parameters indicated that T7(-) and RT preferentially incorporated dTTP opposite O(6)-MeG and O(6)-BzG. The incorporation efficiencies (k(cat)/K(m)) were less for O(6)-BzG than O(6)-MeG for both dCTP and dTTP insertion. Pre-steady-state analysis indicated that the product formed during the burst phase, i.e., the burst amplitude, differed significantly between the unmodified 24-mer/36-G-mer and the O(6)-alkylG-containing substrates. Extension of the O(6)-BzG-containing duplexes was much more difficult for both polymerases as compared to O(6)-MeG, except when RT easily extended the O(6)-BzG:T base pair. The for binding of dCTP or dTTP to a RT*DNA complex containing O(6)-MeG was 8-fold greater than for dNTP binding to a complex containing unmodified DNA. The for a RT*DNA complex containing O(6)-BzG was 50-fold greater. In conclusion, the bulkier O(6)-BzG is a greater block to polymerization by T7(-) and RT than is O(6)-MeG, but some polymerization does occur with an O(6)-BzG substrate. Pre-steady-state analysis indicates that neither dCTP nor dTTP insertion is strongly preferred during polymerization of O(6)-BzG-containing DNA, unlike the case of O(6)-MeG. These results and others regarding polymerase stalling opposite O(6)-MeG and O(6)-BzG are discussed in the following paper in this issue [Woodside, A. M., and Guengerich, F. P. (2002) Biochemistry 41, 1039-1050].

The structural basis for the mutagenicity of O 6 -methyl-guanine lesions

“…The cytotoxic effects of DNA-methylating agents have been exploited in their use as potent anticancer agents. O 6 -methyl-guanine (O6MeG) is mutagenic because polymerases frequently misinsert T opposite O6MeG instead of C, both in vivo (9, 10) and in vitro (11)(12)(13). In this study, we present the crystal structures of complexes of a high-fidelity DNA polymerase with substrates representing several steps of nucleotide insertion opposite O6MeG.…”

“…Phage T7 polymerase shows 170-and 35-fold reductions in efficiency for nucleotide insertion and extension, respectively, as well as a preference for T misinsertion (11). In all polymerases studied to date, incorporation and extension for C and T⅐O6MeG base pairs is more efficient than for of any of the 12 possible unmodified base-base mismatches (11)(12)(13)15).High-fidelity DNA polymerases copy their templates rapidly and accurately. Structures of Bacillus stearothermophilus DNA polymerase I large fragment (BF) as it replicates undamaged (16-18) and damaged (19-21) DNA substrates yield a detailed picture of the structural mechanisms that ensure fidelity at each step of the reaction.…”

“…Phage T7 polymerase shows 170-and 35-fold reductions in efficiency for nucleotide insertion and extension, respectively, as well as a preference for T misinsertion (11). In all polymerases studied to date, incorporation and extension for C and T⅐O6MeG base pairs is more efficient than for of any of the 12 possible unmodified base-base mismatches (11)(12)(13)15).…”

“…High-fidelity polymerases such as exonuclease-deficient E. coli polymerase I (Klenow fragment) (13) or bacteriophage T7 DNA polymerase (11) show an Ϸ7-fold preference for misinsertion of T. By comparison, these polymerases usually show a several thousand-fold preference for insertion of a correct base-pairing partner when copying a normal, undamaged DNA. O6MeG lesions slow the rate of DNA synthesis at the nucleotide incorporation step opposite the lesion (11)(12)(13) and subsequent extension beyond the O6MeG-pair (11,12,14). These kinetic effects vary with polymerase.…”

Kinetics of Nucleotide Incorporation Opposite DNA Bulky Guanine N2 Adducts by Processive Bacteriophage T7 DNA Polymerase (Exonuclease–) and HIV-1 Reverse Transcriptase

“…Because the DNA⅐enzyme dissociation rate is similar for the various adducts (Table V), the thio elemental effect should primarily reflect the rate-limiting contribution of phosphodiester bond formation. The observed elemental effects were generally moderate in these studies (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16) (Table VII). One exception was the dG-CA-S adduct, in which the elemental effect was ϳ100 (Fig.…”