Ribonucleotides and 2'-deoxyribonucleotides are the basic units for RNA and DNA, respectively, and the only difference is the extra 2'-OH group on the ribonucleotide sugar. Cellular rNTP concentrations are much higher than those of dNTP. When copying DNA, DNA polymerases not only select the base of the incoming dNTP to form a Watson-Crick pair with the template base but also distinguish the sugar moiety. Some DNA polymerases use a steric gate residue to prevent rNTP incorporation by creating a clash with the 2'-OH group. Y-family human DNA polymerase eta (hpol eta) is of interest owing to its spacious active site (especially in the major groove) and tolerance of DNA lesions. Here, we show that hpol eta maintains base selectivity when incorporating rNTPs opposite undamaged DNA and the DNA lesions 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxodG) and cyclobutane pyrimidine dimer (CPD) but with rates that are 103-fold lower than for inserting the corresponding dNTPs. X-ray crystal structures show that hpol eta scaffolds the incoming rNTP to pair with the template base (dG) or 8-oxodG with a significant propeller twist. As a result, the 2'-OH group avoids a clash with the steric gate, Phe-18, but the distance between primer end and Pa of the incoming rNTP increases by 1 Å, elevating the energy barrier and slowing polymerization compared with dNTP. In addition, Tyr-92 was identified as a second line of defense to maintain the position of Phe-18.

Mechanism of ribonucleotide incorporation by human DNA polymerase Eta

254th National Meeting (2017)

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