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Incorporation of nucleoside probes opposite O?-methylguanine by Sulfolobus solfataricus DNA polymerase Dpo4: importance of hydrogen bonding.

ABSTRACT: O?-Methylguanine (O?-MeG) is a mutagenic DNA lesion, arising from the action of methylating agents on guanine (G) in DNA. Dpo4, an archaeal low-fidelity Y-family DNA polymerase involved in translesion DNA synthesis (TLS), is a model for studying how human Y-family polymerases bypass DNA adducts. Previous work showed that Dpo4-mediated dTTP incorporation is favored opposite O?-MeG rather than opposite G. However, factors influencing the preference of Dpo4 to incorporate dTTP opposite O?-MeG are not fully defined. In this study, we investigated the influence of structural features of incoming dNTPs on their enzymatic incorporation opposite O?-MeG in a DNA template. To this end, we utilized a new fluorescence-based primer extension assay to evaluate the incorporation efficiency of a panel of synthetic dNTPs opposite G or O?-MeG by Dpo4. In single-dNTP primer extension studies, the synthetic dNTPs were preferentially incorporated opposite G, relative to O?-MeG. Moreover, pyrimidine-based dNTPs were generally better incorporated than purine-based syn-conformation dNTPs. The results suggest that hydrophobicity of the incoming dNTP appears to have little influence on the process of nucleotide selection by Dpo4, with hydrogen bonding capacity being a major influence. Additionally, modifications at the C2-position of dCTP increase the selectivity for incorporation opposite O?-MeG without a significant loss of efficiency.

SUBMITTER: Stornetta A

PROVIDER: S-EPMC3896954 | biostudies-literature | 2013 Sep

REPOSITORIES: biostudies-literature

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Incorporation of nucleoside probes opposite O⁶-methylguanine by Sulfolobus solfataricus DNA polymerase Dpo4: importance of hydrogen bonding.

Stornetta Alessia A Angelov Todor T Guengerich F Peter FP Sturla Shana J SJ

Chembiochem : a European journal of chemical biology 20130819 13

O⁶-Methylguanine (O⁶-MeG) is a mutagenic DNA lesion, arising from the action of methylating agents on guanine (G) in DNA. Dpo4, an archaeal low-fidelity Y-family DNA polymerase involved in translesion DNA synthesis (TLS), is a model for studying how human Y-family polymerases bypass DNA adducts. Previous work showed that Dpo4-mediated dTTP incorporation is favored opposite O⁶-MeG rather than opposite G. However, factors influencing the preference of Dpo4 to incorporate dTTP opposite O⁶-MeG are n ...[more]

PMID: 23959784

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Project description:Translesion DNA polymerases are more efficient at bypass of many DNA adducts than replicative polymerases. Previous work with the translesion polymerase Sulfolobus solfataricus Dpo4 showed a decrease in catalytic efficiency during bypass of bulky N(2)-alkyl guanine (G) adducts with N(2)-isobutylG showing the largest effect, decreasing approximately 120-fold relative to unmodified deoxyguanosine (Zhang, H., Eoff, R. L., Egli, M., Guengerich, F. P. Versatility of Y-family Sulfolobus solfataricus DNA polymerase Dpo4 in translation synthesis past bulky N(2)-alkylguanine adducts. J. Biol. Chem. 2009; 284: 3563-3576). The effect of adduct size on individual catalytic steps has not been easy to decipher because of the difficulty of distinguishing early noncovalent steps from phosphodiester bond formation. We developed a mutant with a single Trp (T239W) to monitor fluorescence changes associated with a conformational change that occurs after binding a correct 2'-deoxyribonucleoside triphosphate (Beckman, J. W., Wang, Q., Guengerich, F. P. Kinetic analysis of nucleotide insertion by a Y-family DNA polymerase reveals conformational change both prior to and following phosphodiester bond formation as detected by tryptophan fluorescence. J. Biol. Chem. 2008; 283: 36711-36723) and, in the present work, utilized this approach to monitor insertion opposite N(2)-alkylG-modified oligonucleotides. We estimated maximal rates for the forward conformational step, which coupled with measured rates of product formation yielded rate constants for the conformational step (both directions) during insertion opposite several N(2)-alkylG adducts. With the smaller N(2)-alkylG adducts, the conformational rate constants were not changed dramatically (<3-fold), indicating that the more sensitive steps are phosphodiester bond formation and partitioning into inactive complexes. With the larger adducts (>or=(2-naphthyl)methyl), the absence of fluorescence changes suggests impaired ability to undergo an appropriate conformational change, consistent with previous structural work.

Dataset Information

Incorporation of nucleoside probes opposite O?-methylguanine by Sulfolobus solfataricus DNA polymerase Dpo4: importance of hydrogen bonding.

Publications

Incorporation of nucleoside probes opposite O⁶-methylguanine by Sulfolobus solfataricus DNA polymerase Dpo4: importance of hydrogen bonding.

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