The Werner syndrome protein limits the error-prone 8-oxo-dG lesion bypass activity of human DNA polymerase kappa.
Bottom Line: 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.
Affiliation: Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR 72205-7199, USA.Show MeSH
Related in: MedlinePlus
Mentions: Pre-steady-state experiments were performed under enzyme-limiting concentrations with varying concentrations of either dCTP or dATP (Figure 3). The observed rate constant (kobs) was plotted as a function of dNTP concentration in order to determine kpol and Kd,dNTP for both error-prone and error-free insertion across from 8-oxo-dG. The addition of WRN stimulates kpol for hpol κ-catalyzed insertion of dCMP opposite 8-oxo-dG 6-fold but does not greatly alter the affinity of the incoming nucleotide (Figure 3A and Table 4). The efficiency of the reaction (kpol/Kd,dNTP) is increased 4-fold for dCMP insertion opposite 8-oxo-dG. The addition of WRN does not alter the efficiency of dAMP insertion by hpol κ (Figure 3B). The overall efficiency of the mis-insertion reaction is essentially the same whether WRN is present or not (Table 4). From these results, we conclude that WRN stimulates the rate constant kpol value for hpol κ-catalyzed insertion of dCMP opposite 8-oxo-dG.
Affiliation: Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR 72205-7199, USA.