Mutagenic conformation of 8-oxo-7,8-dihydro-2′-dGTP in the confines of a DNA polymerase active site

Laboratory of Structural Biology, National Institute of Environmental Health Sciences, US National Institutes of Health, Research Triangle Park, North Carolina, USA.
Nature Structural & Molecular Biology (Impact Factor: 13.31). 07/2010; 17(7):889-90. DOI: 10.1038/nsmb.1852
Source: PubMed


The major product of oxidative base damage is 8-oxo-7,8-dihydro-2'-deoxyguanine (8odG). The coding potential of this lesion is modulated by its glycosidic torsion angle that controls whether its Watson-Crick or Hoogsteen edge is used for base pairing. The 2.0-A structure of DNA polymerase (pol) beta bound with 8odGTP opposite template adenine indicates that the modified nucleotide assumes the mutagenic syn conformation and that the nonmutagenic anti conformation would be incompatible with efficient DNA synthesis.

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Available from: Rajendra Prasad, Sep 29, 2015
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    • "The oxidized nucleotide 8-oxo-dGTP has also dual base-pairing properties, although an intramolecular hydrogen bond between N2 of 8-oxo-dGTP and a non-bridging oxygen on the α-phosphate might strongly favour the syn conformation (29). Moreover, incorporation of 8-oxo-dGTP in the anti conformation seems to be unfavoured due to the steric repulsion between O8 and its sugar-phosphate backbone and also between O8 and the sugar (C2’) of the primer ‘terminus’ (29). Consequently, most DNA polymerases prefer to insert 8-oxo-dGTP opposite a template dA. "
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    ABSTRACT: Full genome sequencing of bacterial genomes has revealed the presence of numerous genes encoding family X DNA polymerases. These enzymes play a variety of biological roles and, accordingly, display often striking functional differences. Here we report that the PolX from the heat-stable organism Thermus thermophilus (TthPolX) inserts the four dNTPs with strong asymmetry. We demonstrate that this behaviour is related to the presence of a single divergent residue in the active site of TthPolX. Mutation of this residue (Ser(266)) to asparagine, the residue present in most PolXs, had a strong effect on TthPolX polymerase activity, increasing and equilibrating the insertion efficiencies of the 4 dNTPs. Moreover, we show that this behaviour correlates with the ability of TthPolX to insert 8-oxo-dGMP. Although the wild-type enzyme inefficiently incorporates 8-oxo-dGMP, the substitution of Ser(266) to asparagine resulted in a dramatic increase in 8-oxo-dGMP incorporation opposite dA. These results suggest that the presence of a serine at position 266 in TthPolX allows the enzyme to minimize the formation of dA:8-oxo-dGMP at the expense of decreasing the insertion rate of pyrimidines. We discuss the structural basis for these effects and the implications of this behaviour for the GO system (BER of 8-oxo-dG lesions).
    Nucleic Acids Research 09/2013; 42(1). DOI:10.1093/nar/gkt870 · 9.11 Impact Factor
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    • "Thus, even with the 160-fold concentration advantage of 8-oxo-dGTP relative to dGTP employed in the mutagenesis procedure, the incorporation of 8-oxo-dGTP likely was disfavored at C positions. The recently reported crystal structure of 8-oxo-dGTP bound opposite dA in the active site DNA polymerase β confirms the substantially more favorable geometry of this pairing compared to that of 8-oxo-dGTP and dC (19). dPTP, in contrast, gave rise to A-to-G and G-to-A transition mutations in a symmetrical manner, occurring at a frequency of 1.7 and 1.6%, respectively. "
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    ABSTRACT: Next-generation DNA sequencing technology was used to score >100,000 mutations resulting from exposure of a nucleic acid template to a mutagenic dNTP analog during a single pass of a DNA polymerase. An RNA template of known secondary structure was reverse transcribed in the presence of 8-oxo-dGTP, dPTP or both, followed by forward transcription in the presence of standard NTPs. Each mutagen, whether used alone or in combination, resulted in a highly characteristic mutation profile. Mutations were generated at a mean frequency of 1-2% per eligible nucleotide position, but there was substantial variation in the frequency of mutation at different positions, with a SD close to the mean. This variation was partly due to the identity of the immediately surrounding nucleotides and was not significantly influenced by the secondary structure of the RNA template. Most of the variation appears to result from idiosyncratic features that derive from local sequence context, demonstrating how different genetic sequences have different chemical phenotypes.
    Nucleic Acids Research 12/2010; 38(22):8095-104. DOI:10.1093/nar/gkq685 · 9.11 Impact Factor
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