title: Human Translesion Polymerase κ Exhibits Enhanced Activity and Reduced Fidelity Two Nucleotides from G-Quadruplex DNA.
abstract: We have investigated the in vitro properties of human Y-family polymerase κ (hpol κ) on G-quadruplex DNA (G4 DNA). Similar to hpol η, another Y-family member implicated in replication of G4 motifs, hpol κ 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 κ than they do to that by hpol η. However, kinetic analysis revealed that hpol κ activity increases considerably when the enzyme is 2-3 nucleotides from the G4 motif, a trend that was reported previously for hpol η, though the increase was less pronounced. The increase in hpol κ 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 κ when the counterion was either potassium or sodium but not in the presence of lithium. The activity of hpol κ 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 η, but not for the B-family member hpol ε, 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 κ activity was enhanced relative to those of other Y-family members when the enzyme is 2-3 nucleotides from the G4 motif, but hpol κ appears to be less competent than hpol η at copying tetrad-associated guanines.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences , Little Rock, Arkansas 72205-7199, United States.
authors.forename: Sarah
authors.initials: S
authors.lastname: Eddy
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences , Little Rock, Arkansas 72205-7199, United States.
authors.forename: Magdalena
authors.initials: M
authors.lastname: Tillman
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences , Little Rock, Arkansas 72205-7199, United States.
authors.forename: Leena
authors.initials: L
authors.lastname: Maddukuri
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences , Little Rock, Arkansas 72205-7199, United States.
authors.forename: Amit
authors.initials: A
authors.lastname: Ketkar
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences , Little Rock, Arkansas 72205-7199, United States.
authors.forename: Maroof K
authors.initials: MK
authors.lastname: Zafar
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences , Little Rock, Arkansas 72205-7199, United States.
authors.forename: Robert L
authors.initials: RL
authors.lastname: Eoff
mesh_terms: D004247:DNA / Q000378:metabolism*; D004249:DNA Damage; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D005454:Fluorescence Polarization; D054856:G-Quadruplexes; D006801:Humans; D007700:Kinetics; D000099270:Y-Family DNA Polymerases
chemical_list: D004247:DNA; D004259:DNA-Directed DNA Polymerase; C404302:POLK protein, human; D000099270:Y-Family DNA Polymerases