Human Translesion Polymerase κ Exhibits Enhanced Activity and Reduced Fidelity Two Nucleotides from G-Quadruplex DNA

We have investigated the in vitro properties of human Y-family polymerase kappa (hpol kappa) on G-quadruplex DNA (G4 DNA). Similar to hpol eta, another Y-family member implicated in replication of G4 motifs, hpol kappa bound G4 DNA with a 5.7-fold preference over control, non-G4 DNA. Results from pol extension assays are consistent with the notion that G-quadruplexes present a stronger barrier to DNA synthesis by hpol kappa than they do to that by hpol eta. However, kinetic analysis revealed that hpol kappa activity increases considerably when the enzyme is 2-3 nucleotides from the G4 motif, a trend that was reported previously for hpol eta, though the increase was less pronounced. The increase in hpol kappa activity on G4 DNA was readily observed in the presence of either potassium or sodium but much less so when lithium was used in the buffer. The increased activity 2-3 nucleotides from the G4 motif was accompanied by a decrease in the fidelity of hpol kappa when the counterion was either potassium or sodium but not in the presence of lithium. The activity of hpol kappa decreased progressively as the primer was moved closer than 2 nucleotides from the G4 motif when either potassium or sodium was used to stabilize the G-quadruplex. Interestingly, the decrease in catalytic activity at the site of the quadruplex observed in potassium-containing buffer was accompanied by an increase in fidelity on G4 substrates versus control non-G4 substrates. This trend of increased fidelity in copying a tetrad associated guanine was observed previously for hpol eta, but not for the B-family member hpol epsilon, which exhibited a large decrease in both efficiency and fidelity in the attempt to copy the first guanine in the G4 motif. In summary, hpol kappa activity was enhanced relative to those of other Y-family members when the enzyme is 2-3 nucleotides from the G4 motif, but hpol kappa appears to be less competent than hpol eta at copying tetrad-associated guanines.