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DNA polymerases insert dATP opposite the oxidative damage product 7,8-dihydro-8-oxodeoxyguanosine (8-oxoG) instead of dCTP, to the extent of >90% with some polymerases. Steady-state kinetics with the Y-family Sulfolobus solfataricus DNA polymerase IV (Dpo4) showed 90-fold higher incorporation efficiency of dCTP > dATP opposite 8-oxoG and 4-fold higher efficiency of extension beyond an 8-oxoG:C pair than an 8-oxoG:A pair. The catalytic efficiency for these events (with dCTP or C) was similar for G and 8-oxoG templates. Mass spectral analysis of extended DNA primers showed 295% incorporation of dCTP > dATP opposite 8-oxoG. Pre-steady-state kinetics showed faster rates of dCTP incorporation opposite 8-oxoG than G. The measured Kd,dCTP was 15-fold lower for an oligonucleotide containing 8-oxoG than with G. Extension beyond an 8-oxoG:C pair was similar to G:C and faster than for an 8-oxoG:A pair, in contrast to other polymerases. The Ea for dCTP insertion opposite 8-oxoG was lower than for opposite G. Crystal structures of Dpo4 complexes with oligonucleotides were solved with C, A, and G nucleoside triphosphates placed opposite 8-oxoG. With ddCTP, dCTP, and dATP the phosphodiester bonds were formed even in the presence of Ca2+. The 8-oxoG:C pair showed classic Watson-Crick geometry; the 8-oxoG:A pair was in the syn:anti configuration, with the A hybridized in a Hoogsteen pair with 8-oxoG. With dGTP placed opposite 8-oxoG, pairing was not to the 8-oxoG but to the 5′ C (and in classic Watson-Crick geometry), consistent with the low frequency of this frameshift event observed in the catalytic assays. [ABSTRACT FROM AUTHOR]

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 alkyl-transferase (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 ∼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 OS-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-MeG and O6-BzG and AGT but not with a Gly mutation at Arg12s, 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. [ABSTRACT FROM AUTHOR]

1,N²-Etheno(∈)guanine is a mutagenic DNA lesion derived from lipid oxidation products and also from some chemical carcinogens. Gel electrophoretic analysis of the products of primer extension by Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4) indicated preferential incorporation of A opposite 3′-(1,N²-∈-G)TACT-5′, among the four dNTPs tested individually. With the template 3′-(1,N²-∈-G)CACT-5′, both G and A were incorporated. When primer extension was done in the presence of a mixture of all four dNTPs, high pressure liquid chromatography-mass spectrometry analysis of the products indicated that (opposite 3′-(1,N²-∈-G)CACT-5′) the major product was 5′-GTGA-3′ and the minor product was 5′-AGTGA-3′. With the template 3′.(1,N²-∈-G)TACT-5′, the following four products were identified by high pressure liquid chromatography-mass spectrometry: 5′-AATGA-3′, 5′-ATTGA-3′, 5′-ATGA-3′, and 5′-TGA-3′. An x-ray crystal structure of Dpo4 was solved (2.1 Å) with a primer-template and A placed in the primer to be opposite the 1,N²-∈-G in the template 3′-(1,N²-∈-G)TACT 5′. The added A in the primer was paired across the template T with classic Watson-Crick geometry. Similar structures were observed in a ternary Dpo4-DNA-dATP complex and a ternary Dpo4-DNA-ddATP complex, with d(d)ATP opposite the template T. A similar structure was observed with a ddGTP adjacent to the primer and opposite the C next to 1,N²-∈-G in 3′-(1,N²-∈-G)CACT-5′. We concluded that Dpo4 uses several mechanisms, including A incorporation opposite 1,N²-∈-G and also a variation of dNTP-stabilized misalignment, to generate both base pair and frameshift mutations. [ABSTRACT FROM AUTHOR]

Six oligonucleotides with carcinogen derivatives bound at the N2 atom of deoxyguanosine were prepared, including adducts derived from butadiene, acrolein, crotonaldehyde, and styrene, and examined for effects on the replicative enzymes bacteriophage DNA polymerase T7- (T7-) and HIV-1 reverse transcriptase for comparison with previous work on smaller DNA adducts. All of these adducts strongly blocked dCTP incorporation opposite the adducts, dATP was preferentially incorporated opposite the acrolein and crotonaldehyde adducts, and dTTP incorporation was preferred at the butadiene- and styrene-derived adducts. Steady-state kinetic analysis indicated that the reduced catalytic efficiency with adducted DNA involved both an increased Km and attenuated kcat. Fluorescence estimates of Kd and pre-steady-state kinetic measurements of koff showed no significantly decreased affinity of T7- with the adducted oligonucleotides or the dNTP. Pre-steady-state kinetics showed no burst phase kinetics for dNTP incorporation with any of the modified oligonucleotides. These results indicate that phosphodiester bond formation or a conformational change of the enzyme. DNA complex is rate-limiting instead of the step involving release of the oligonucleotide. Thio elemental effects for dNTP incorporation were generally relatively small but variable, indicating that the presence of adducts may sometimes make phosphodiester bond formation rate-limiting but not always. [ABSTRACT FROM AUTHOR]

A correction to the article "Efficient and High Fidelity Incorporation of dCTP Opposite 7, 8-Dihydro-8-Oxodeoxyguanosine by Sulfolobus Solfataricus DNA Polymerase Dpo4," in the volume 281, 2006 issue is presented.

Previous studies have shown that replicative bacterial and viral DNA polymerases are able to bypass the mutagenic lesions O6-methyl and -benzyl (Bz) G. Recombinant human polymerase (pol) δ also copied past these two lesions but was totally blocked by O6-[4-oxo-4-(3-pyridyl)butyl] (Pob)G, an important mutagenic lesion formed following metabolic activation of the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone. The human translesion pols ɩ and κ produced mainly only 1-base incorporation opposite O6-MeG and O6-BzG and had very low activity in copying O6-PobG. Human pol η copied past all three adducts. Steady-state kinetic analysis showed similar efficiencies of insertion opposite the O6-alkylG adducts for dCTP and dTTP with pol η and κ pol ɩ showed a strong preference for dTTP. pot η, ɩ, and κ showed pre-steady-state kinetic bursts for dCTP incorporation opposite G and O6-MeG but little, if any, for O6-BzG or O6-PobG. Analysis of the pol η O6-PobG products indicated that the insertion of G was opposite the base (C) 5′ of the adduct, but this product was not extended. Mass spectrometry analysis of all of the pol η primer extension products indicated multiple components, mainly with C or T inserted opposite O6-alkylG but with no deletions in the cases of O6-MeG and O6-PobG. With pol η and O6-BzG, products were also obtained with –1 and –2 deletions and also with A inserted (opposite O6-BzG). The results with pol η may be relevant to some mutations previously reported with O6-alkylG adducts in mammalian cells. [ABSTRACT FROM AUTHOR]