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
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Mentions: WRN is a protein with several enzymatic functions and multiple domains (Figure 1A) (48). We began our study by testing the ability of full-length WRN and a series of WRN truncation mutants to stimulate extension by hpol κ on unmodified DNA templates (Figure 1B). All WRN stocks were checked for contaminating polymerase activity by performing extension experiments out to an hour in the absence of hpol κ (data not shown). Extension assays were repeated at least twice to ensure the reproducibility of any stimulation of polymerization by WRN. Total product formed in each lane was plotted as a function of time and the resulting curve was fit to a single-exponential equation to obtain an estimate of the rate constant for primer extension by hpol κ. To compare the relative effect of different WRN constructs on hpol κ extension activity, we divided the rate constant for primer extension in the presence of WRN by the rate constant for primer extension when hpol κ is alone and multiplying by a factor of one hundred to yield percent activity. In this way, we were able to make quantitative comparisons for stimulation of pol extension activity by different WRN constructs.
Affiliation: Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR 72205-7199, USA.