X-Pol Biotech S.L.U. Parque Científico de Madrid. Cantoblanco, Madrid 28049, Spain, Department of Pharmacological Sciences, Stony Brook University, Stony Brook, NY 11794, USA and Centro de Biología Molecular Severo Ochoa (CSIC-UAM). Cantoblanco, Madrid 28049, Spain.
Nucleic Acids Res. 2014 Jan;42(1):534-43. doi: 10.1093/nar/gkt870. Epub 2013 Sep 30.
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).
全基因组测序揭示了许多编码 X 家族 DNA 聚合酶的基因的存在。这些酶具有多种生物学功能，因此表现出显著的功能差异。本文报道了耐热生物 Thermus thermophilus（TthPolX）中的 PolX 以强烈的不对称性插入四种 dNTP。我们证明这种行为与 TthPolX 活性位点中单个独特残基的存在有关。该残基（Ser266）突变为大多数 PolX 中存在的天冬酰胺，对 TthPolX 聚合酶活性有强烈影响，增加并平衡了 4 种 dNTP 的插入效率。此外，我们表明这种行为与 TthPolX 插入 8-oxo-dGMP 的能力相关。尽管野生型酶对 8-oxo-dGMP 的掺入效率较低，但将 Ser266 突变为天冬酰胺导致在 dA 对面掺入 8-oxo-dGMP 的能力急剧增加。这些结果表明，TthPolX 中 266 位丝氨酸的存在允许酶以牺牲嘧啶插入率为代价，最小化 dA：8-oxo-dGMP 的形成。我们讨论了这些效应的结构基础以及这种行为对 GO 系统（8-oxo-dG 损伤的 BER）的影响。
