Limits...
The Werner syndrome protein limits the error-prone 8-oxo-dG lesion bypass activity of human DNA polymerase kappa.

Maddukuri L, Ketkar A, Eddy S, Zafar MK, Eoff RL - Nucleic Acids Res. (2014)

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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR 72205-7199, USA.

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WRN stimulates the pre-steady-state rate constant kpol for dCMP insertion opposite 8-oxo-dG by hpol κ. (A) hPol κ-catalyzed (25 nM) incorporation of dCTP opposite 8-oxo-dG-containing p/t-DNA (50 nM) was performed in the presence of WRN1-1432 (250 nM) and varying concentrations of dCTP (0.5-1000 μM). The product curves were fit to Equation (2) except for the experiment with 0.5 μM, which was fit to Equation (1). The observed rate of product formation (kobs) was plotted as a function of [dCTP] for experiments with hpol κ alone (•) and for experiments with hpol κ performed in the presence of WRN1-1432. The resulting kinetic parameters are listed in Table 4. (B) hPol κ-catalyzed (25 nM) incorporation of dATP opposite 8-oxo-dG-containing p/t-DNA (50 nM) was performed in the presence of WRN1-1432 (250 nM) and varying concentrations of dATP (0.5-500 μM). The product curves were fit to Equation (2). The observed rate of product formation (kobs) was plotted as a function of [dATP] for experiments with hpol κ alone (•) and for experiments with hpol κ performed in the presence of WRN1-1432. The resulting kinetic parameters are listed in Table 4.
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Figure 3: WRN stimulates the pre-steady-state rate constant kpol for dCMP insertion opposite 8-oxo-dG by hpol κ. (A) hPol κ-catalyzed (25 nM) incorporation of dCTP opposite 8-oxo-dG-containing p/t-DNA (50 nM) was performed in the presence of WRN1-1432 (250 nM) and varying concentrations of dCTP (0.5-1000 μM). The product curves were fit to Equation (2) except for the experiment with 0.5 μM, which was fit to Equation (1). The observed rate of product formation (kobs) was plotted as a function of [dCTP] for experiments with hpol κ alone (•) and for experiments with hpol κ performed in the presence of WRN1-1432. The resulting kinetic parameters are listed in Table 4. (B) hPol κ-catalyzed (25 nM) incorporation of dATP opposite 8-oxo-dG-containing p/t-DNA (50 nM) was performed in the presence of WRN1-1432 (250 nM) and varying concentrations of dATP (0.5-500 μM). The product curves were fit to Equation (2). The observed rate of product formation (kobs) was plotted as a function of [dATP] for experiments with hpol κ alone (•) and for experiments with hpol κ performed in the presence of WRN1-1432. The resulting kinetic parameters are listed in Table 4.

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.


The Werner syndrome protein limits the error-prone 8-oxo-dG lesion bypass activity of human DNA polymerase kappa.

Maddukuri L, Ketkar A, Eddy S, Zafar MK, Eoff RL - Nucleic Acids Res. (2014)

WRN stimulates the pre-steady-state rate constant kpol for dCMP insertion opposite 8-oxo-dG by hpol κ. (A) hPol κ-catalyzed (25 nM) incorporation of dCTP opposite 8-oxo-dG-containing p/t-DNA (50 nM) was performed in the presence of WRN1-1432 (250 nM) and varying concentrations of dCTP (0.5-1000 μM). The product curves were fit to Equation (2) except for the experiment with 0.5 μM, which was fit to Equation (1). The observed rate of product formation (kobs) was plotted as a function of [dCTP] for experiments with hpol κ alone (•) and for experiments with hpol κ performed in the presence of WRN1-1432. The resulting kinetic parameters are listed in Table 4. (B) hPol κ-catalyzed (25 nM) incorporation of dATP opposite 8-oxo-dG-containing p/t-DNA (50 nM) was performed in the presence of WRN1-1432 (250 nM) and varying concentrations of dATP (0.5-500 μM). The product curves were fit to Equation (2). The observed rate of product formation (kobs) was plotted as a function of [dATP] for experiments with hpol κ alone (•) and for experiments with hpol κ performed in the presence of WRN1-1432. The resulting kinetic parameters are listed in Table 4.
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Figure 3: WRN stimulates the pre-steady-state rate constant kpol for dCMP insertion opposite 8-oxo-dG by hpol κ. (A) hPol κ-catalyzed (25 nM) incorporation of dCTP opposite 8-oxo-dG-containing p/t-DNA (50 nM) was performed in the presence of WRN1-1432 (250 nM) and varying concentrations of dCTP (0.5-1000 μM). The product curves were fit to Equation (2) except for the experiment with 0.5 μM, which was fit to Equation (1). The observed rate of product formation (kobs) was plotted as a function of [dCTP] for experiments with hpol κ alone (•) and for experiments with hpol κ performed in the presence of WRN1-1432. The resulting kinetic parameters are listed in Table 4. (B) hPol κ-catalyzed (25 nM) incorporation of dATP opposite 8-oxo-dG-containing p/t-DNA (50 nM) was performed in the presence of WRN1-1432 (250 nM) and varying concentrations of dATP (0.5-500 μM). The product curves were fit to Equation (2). The observed rate of product formation (kobs) was plotted as a function of [dATP] for experiments with hpol κ alone (•) and for experiments with hpol κ performed in the presence of WRN1-1432. The resulting kinetic parameters are listed in Table 4.
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.

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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR 72205-7199, USA.

Show MeSH
Related in: MedlinePlus