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Structure-function relationships in miscoding by Sulfolobus solfataricus DNA polymerase Dpo4: guanine N2,N2-dimethyl substitution produces inactive and miscoding polymerase complexes.

ABSTRACT: Previous work has shown that Y-family DNA polymerases tolerate large DNA adducts, but a substantial decrease in catalytic efficiency and fidelity occurs during bypass of N2,N2-dimethyl (Me2)-substituted guanine (N2,N2-Me2G), in contrast to a single methyl substitution. Therefore, it is unclear why the addition of two methyl groups is so disruptive. The presence of N2,N2-Me2G lowered the catalytic efficiency of the model enzyme Sulfolobus solfataricus Dpo4 16,000-fold. Dpo4 inserted dNTPs almost at random during bypass of N2,N2-Me2G, and much of the enzyme was kinetically trapped by an inactive ternary complex when N2,N2-Me2G was present, as judged by a reduced burst amplitude (5% of total enzyme) and kinetic modeling. One crystal structure of Dpo4 with a primer having a 3'-terminal dideoxycytosine (Cdd) opposite template N2,N2-Me2G in a post-insertion position showed Cdd folded back into the minor groove, as a catalytically incompetent complex. A second crystal had two unique orientations for the primer terminal Cdd as follows: (i) flipped into the minor groove and (ii) a long pairing with N2,N2-Me2G in which one hydrogen bond exists between the O-2 atom of Cdd and the N-1 atom of N2,N2-Me2G, with a second water-mediated hydrogen bond between the N-3 atom of Cdd and the O-6 atom of N2,N2-Me2G. A crystal structure of Dpo4 with dTTP opposite template N2,N2-Me2G revealed a wobble orientation. Collectively, these results explain, in a detailed manner, the basis for the reduced efficiency and fidelity of Dpo4-catalyzed bypass of N2,N2-Me2G compared with mono-substituted N2-alkyl G adducts.

SUBMITTER: Zhang H

PROVIDER: S-EPMC2719408 | biostudies-literature | 2009 Jun

REPOSITORIES: biostudies-literature

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Structure-function relationships in miscoding by Sulfolobus solfataricus DNA polymerase Dpo4: guanine N2,N2-dimethyl substitution produces inactive and miscoding polymerase complexes.

Zhang Huidong H Eoff Robert L RL Kozekov Ivan D ID Rizzo Carmelo J CJ Egli Martin M Guengerich F Peter FP

The Journal of biological chemistry 20090601 26

Previous work has shown that Y-family DNA polymerases tolerate large DNA adducts, but a substantial decrease in catalytic efficiency and fidelity occurs during bypass of N2,N2-dimethyl (Me2)-substituted guanine (N2,N2-Me2G), in contrast to a single methyl substitution. Therefore, it is unclear why the addition of two methyl groups is so disruptive. The presence of N2,N2-Me2G lowered the catalytic efficiency of the model enzyme Sulfolobus solfataricus Dpo4 16,000-fold. Dpo4 inserted dNTPs almost ...[more]

PMID: 19542237

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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.

Project description:Oligonucleotides were synthesized containing the 7-(2-oxoheptyl)-etheno-dGuo adduct, which is derived from the reaction of dGuo and the lipid peroxidation product 4-oxo-2-nonenal. The in vitro replication of 7-(2-oxoheptyl)-etheno-dGuo by the model Y-family polymerase Sulfolobus solfataricus P2 DNA Polymerase IV (Dpo4) was examined in two sequences. The extension products were sequenced using an improved LC-ESI-MS/MS protocol developed in our laboratories, and the results were compared to that of the 1,N(2)-etheno-dGuo adduct in the same sequence contexts. Both etheno adducts were highly miscoding when situated in 5'-TXG-3' local sequence contexts with <4% of the extension products being derived from error-free bypass. The major extension products resulted from the misinsertion of Ade opposite the adduct and a one-base deletion. The major extension products from replication of the etheno lesions in a 5'-CXG-3' local sequence context were the result of misinsertion of Ade, a one-base deletion, and error-free bypass. Other minor extension products were also identified. The 7-(2-oxoheptyl)-etheno-dGuo lesion resulted in a larger frequency of misinsertion of Ade, whereas the 1,N(2)-etheno-dGuo gave more of the one-base deletion product. Conformational studies of duplex DNA containing the 7-(2-oxoheptyl)-etheno-dGuo in a 5'-TXG-3' sequence context by NMR indicated the presence of a pH-dependent conformational transition, likely involving the glycosyl bond at the adducted guanosine; the pK(a) for this transition was lower than that observed for the 1,N(2)-epsilon-dGuo lesion. However, the 7-(2-oxoheptyl)-etheno-dGuo lesion, the complementary Cyt, and both flanking base pairs remained disordered at all pH values, which is attributed to the presence of the hydrophobic heptyl group of the 7-(2-oxoheptyl)-etheno-dGuo lesion. The altered pK(a) value and the structural disorder at the 7-(2-oxoheptyl)-etheno-dGuo lesion site, as compared to the same sequence containing the 1,N(2)-etheno-dGuo, may contribute to higher frequency of misinsertion of Ade.

Dataset Information

Structure-function relationships in miscoding by Sulfolobus solfataricus DNA polymerase Dpo4: guanine N2,N2-dimethyl substitution produces inactive and miscoding polymerase complexes.

Publications

Structure-function relationships in miscoding by Sulfolobus solfataricus DNA polymerase Dpo4: guanine N2,N2-dimethyl substitution produces inactive and miscoding polymerase complexes.

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