title: The Werner syndrome protein limits the error-prone 8-oxo-dG lesion bypass activity of human DNA polymerase kappa.
abstract: Human DNA polymerase kappa (hpol κ) is the only Y-family member to preferentially insert dAMP opposite 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxo-dG) during translesion DNA synthesis. We have studied the mechanism of action by which hpol κ activity is modulated by the Werner syndrome protein (WRN), a RecQ helicase known to influence repair of 8-oxo-dG. Here we show that WRN stimulates the 8-oxo-dG bypass activity of hpol κ in vitro by enhancing the correct base insertion opposite the lesion, as well as extension from dC:8-oxo-dG base pairs. Steady-state kinetic analysis reveals that WRN improves hpol κ-catalyzed dCMP insertion opposite 8-oxo-dG ∼10-fold and extension from dC:8-oxo-dG by 2.4-fold. Stimulation is primarily due to an increase in the rate constant for polymerization (kpol), as assessed by pre-steady-state kinetics, and it requires the RecQ C-terminal (RQC) domain. In support of the functional data, recombinant WRN and hpol κ were found to physically interact through the exo and RQC domains of WRN, and co-localization of WRN and hpol κ was observed in human cells treated with hydrogen peroxide. Thus, WRN limits the error-prone bypass of 8-oxo-dG by hpol κ, which could influence the sensitivity to oxidative damage that has previously been observed for Werner's syndrome cells.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR 72205-7199, USA.
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, AR 72205-7199, USA.
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, AR 72205-7199, USA.
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, AR 72205-7199, USA.
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, AR 72205-7199, USA rleoff@uams.edu.
authors.forename: Robert L
authors.initials: RL
authors.lastname: Eoff
mesh_terms: D000080242:8-Hydroxy-2'-Deoxyguanosine; D004247:DNA / Q000096:biosynthesis* / Q000378:metabolism; D018736:DNA Adducts / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D003849:Deoxyguanosine / Q000031:analogs & derivatives* / Q000378:metabolism; D005090:Exodeoxyribonucleases / Q000737:chemistry / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D017434:Protein Structure, Tertiary; D053484:RecQ Helicases / Q000737:chemistry / Q000378:metabolism*; D000099270:Y-Family DNA Polymerases
chemical_list: D000080242:8-Hydroxy-2'-Deoxyguanosine; D004247:DNA; D018736:DNA Adducts; D004259:DNA-Directed DNA Polymerase; D003849:Deoxyguanosine; D005090:Exodeoxyribonucleases; D053484:RecQ Helicases; C404302:POLK protein, human; D000099270:Y-Family DNA Polymerases