The Active Site of TthPolX is Adapted to Prevent 8-oxo-dGTP Misincorporation

Overview

Journal Nucleic Acids Res

Publisher Oxford University Press

Specialty Biochemistry

Date 2013 Oct 3

PMID 24084083

Citations 5

Authors

Patricia Garrido

Edison Mejia

Miguel Garcia-Diaz

Luis Blanco

Angel J Picher

Affiliations

Soon will be listed here.

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).

Citing Articles

Noncanonical prokaryotic X family DNA polymerases lack polymerase activity and act as exonucleases.

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The PHP domain of PolX from Staphylococcus aureus aids high fidelity DNA synthesis through the removal of misincorporated deoxyribo-, ribo- and oxidized nucleotides.

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Insights into the effect of minor groove interactions and metal cofactors on mutagenic replication by human DNA polymerase β.

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Nucleotide binding interactions modulate dNTP selectivity and facilitate 8-oxo-dGTP incorporation by DNA polymerase lambda.

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