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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The WRN exonuclease domain preferentially degrades dA:8-oxo-dG mis-pairs. (A) WRN exo (100 nM) activity was measured for 14/18-mer DNA substrates (200 nM) possessing the terminal bp indicated above the gel image. (B) WRN-catalyzed exonucleolytic degradation of primer was quantified and plotted as a function of time. The sum of all degraded product bands was quantified. The data were analyzed by linear regression to estimate the rate of primer degradation for the linear portion of the velocity curve: dC:dG (closed circles): vobs = 0.33 ± 0.02 nM min−1; dC:8-oxo-dG (open circles): vobs = 0.40 ± 0.01 nM min−1; dA:dG (closed squares): vobs = 0.87 ± 0.04 nM min−1; dA:8-oxo-dG (open squares): vobs = 0.86 ± 0.03 nM min−1. (C) hpol κ (2 nM) extension from 14/18-mer DNA substrates (200 nM) in the presence of WRN exo (100 nM). The reaction was initiated upon addition of dGTP (100 μM) and MgCl2 (5 mM). (D) WRN-catalyzed exonucleolytic degradation of primer in the presence of competing pol activity was quantified and plotted as a function of time. Both pol and exo product bands were quantified and the amount of degraded primer calculated as a fraction of the total DNA in each lane. The data were analyzed by linear regression to estimate the rate of primer degradation for the linear portion of the velocity curve: dC:dG (closed circles): vobs = 0.069 ± 0.009 nM min−1; dC:8-oxo-dG (open circles): vobs = 0.29 ± 0.02 nM min−1; dA:dG (closed squares): vobs = 2.7 ± 0.3 nM min−1; dA:8-oxo-dG (open squares): vobs = 2.0 ± 0.1 nM min−1.
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Figure 4: The WRN exonuclease domain preferentially degrades dA:8-oxo-dG mis-pairs. (A) WRN exo (100 nM) activity was measured for 14/18-mer DNA substrates (200 nM) possessing the terminal bp indicated above the gel image. (B) WRN-catalyzed exonucleolytic degradation of primer was quantified and plotted as a function of time. The sum of all degraded product bands was quantified. The data were analyzed by linear regression to estimate the rate of primer degradation for the linear portion of the velocity curve: dC:dG (closed circles): vobs = 0.33 ± 0.02 nM min−1; dC:8-oxo-dG (open circles): vobs = 0.40 ± 0.01 nM min−1; dA:dG (closed squares): vobs = 0.87 ± 0.04 nM min−1; dA:8-oxo-dG (open squares): vobs = 0.86 ± 0.03 nM min−1. (C) hpol κ (2 nM) extension from 14/18-mer DNA substrates (200 nM) in the presence of WRN exo (100 nM). The reaction was initiated upon addition of dGTP (100 μM) and MgCl2 (5 mM). (D) WRN-catalyzed exonucleolytic degradation of primer in the presence of competing pol activity was quantified and plotted as a function of time. Both pol and exo product bands were quantified and the amount of degraded primer calculated as a fraction of the total DNA in each lane. The data were analyzed by linear regression to estimate the rate of primer degradation for the linear portion of the velocity curve: dC:dG (closed circles): vobs = 0.069 ± 0.009 nM min−1; dC:8-oxo-dG (open circles): vobs = 0.29 ± 0.02 nM min−1; dA:dG (closed squares): vobs = 2.7 ± 0.3 nM min−1; dA:8-oxo-dG (open squares): vobs = 2.0 ± 0.1 nM min−1.

Mentions: Next, we tested whether the isolated WRN exo domain could preferentially degrade 8-oxo-dG containing bp (Figure 4A). In the absence of hpol κ, the WRN1-333 exo domain exhibits a clear preference for the degradation of dA-containing mis-pairs regardless of whether 8-oxo-dG is present or unmodified dG. The rate of degradation was ∼0.9 nM min−1 for the two dA-containing mis-pairs, whereas the rate of degradation for dC:dG and dC:8-oxo-dG bp was 0.33 and 0.40 nM min−1, respectively (Figure 4B). The rate of WRN-catalyzed dA:8-oxo-dG degradation is ∼2-fold faster than dC: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)

The WRN exonuclease domain preferentially degrades dA:8-oxo-dG mis-pairs. (A) WRN exo (100 nM) activity was measured for 14/18-mer DNA substrates (200 nM) possessing the terminal bp indicated above the gel image. (B) WRN-catalyzed exonucleolytic degradation of primer was quantified and plotted as a function of time. The sum of all degraded product bands was quantified. The data were analyzed by linear regression to estimate the rate of primer degradation for the linear portion of the velocity curve: dC:dG (closed circles): vobs = 0.33 ± 0.02 nM min−1; dC:8-oxo-dG (open circles): vobs = 0.40 ± 0.01 nM min−1; dA:dG (closed squares): vobs = 0.87 ± 0.04 nM min−1; dA:8-oxo-dG (open squares): vobs = 0.86 ± 0.03 nM min−1. (C) hpol κ (2 nM) extension from 14/18-mer DNA substrates (200 nM) in the presence of WRN exo (100 nM). The reaction was initiated upon addition of dGTP (100 μM) and MgCl2 (5 mM). (D) WRN-catalyzed exonucleolytic degradation of primer in the presence of competing pol activity was quantified and plotted as a function of time. Both pol and exo product bands were quantified and the amount of degraded primer calculated as a fraction of the total DNA in each lane. The data were analyzed by linear regression to estimate the rate of primer degradation for the linear portion of the velocity curve: dC:dG (closed circles): vobs = 0.069 ± 0.009 nM min−1; dC:8-oxo-dG (open circles): vobs = 0.29 ± 0.02 nM min−1; dA:dG (closed squares): vobs = 2.7 ± 0.3 nM min−1; dA:8-oxo-dG (open squares): vobs = 2.0 ± 0.1 nM min−1.
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Figure 4: The WRN exonuclease domain preferentially degrades dA:8-oxo-dG mis-pairs. (A) WRN exo (100 nM) activity was measured for 14/18-mer DNA substrates (200 nM) possessing the terminal bp indicated above the gel image. (B) WRN-catalyzed exonucleolytic degradation of primer was quantified and plotted as a function of time. The sum of all degraded product bands was quantified. The data were analyzed by linear regression to estimate the rate of primer degradation for the linear portion of the velocity curve: dC:dG (closed circles): vobs = 0.33 ± 0.02 nM min−1; dC:8-oxo-dG (open circles): vobs = 0.40 ± 0.01 nM min−1; dA:dG (closed squares): vobs = 0.87 ± 0.04 nM min−1; dA:8-oxo-dG (open squares): vobs = 0.86 ± 0.03 nM min−1. (C) hpol κ (2 nM) extension from 14/18-mer DNA substrates (200 nM) in the presence of WRN exo (100 nM). The reaction was initiated upon addition of dGTP (100 μM) and MgCl2 (5 mM). (D) WRN-catalyzed exonucleolytic degradation of primer in the presence of competing pol activity was quantified and plotted as a function of time. Both pol and exo product bands were quantified and the amount of degraded primer calculated as a fraction of the total DNA in each lane. The data were analyzed by linear regression to estimate the rate of primer degradation for the linear portion of the velocity curve: dC:dG (closed circles): vobs = 0.069 ± 0.009 nM min−1; dC:8-oxo-dG (open circles): vobs = 0.29 ± 0.02 nM min−1; dA:dG (closed squares): vobs = 2.7 ± 0.3 nM min−1; dA:8-oxo-dG (open squares): vobs = 2.0 ± 0.1 nM min−1.
Mentions: Next, we tested whether the isolated WRN exo domain could preferentially degrade 8-oxo-dG containing bp (Figure 4A). In the absence of hpol κ, the WRN1-333 exo domain exhibits a clear preference for the degradation of dA-containing mis-pairs regardless of whether 8-oxo-dG is present or unmodified dG. The rate of degradation was ∼0.9 nM min−1 for the two dA-containing mis-pairs, whereas the rate of degradation for dC:dG and dC:8-oxo-dG bp was 0.33 and 0.40 nM min−1, respectively (Figure 4B). The rate of WRN-catalyzed dA:8-oxo-dG degradation is ∼2-fold faster than dC: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