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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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Related in: MedlinePlus

WRN stimulates hpol κ19-526 activity on undamaged DNA templates. (A) An overview of the full-length WRN protein showing domains with either structural or catalytic properties relevant to the current study. HRDC: Helicase and RNaseD C-terminal; NLS: nuclear localization signal; PIP: PCNA interacting peptide; RQC: RecQ C-terminal; the E84A mutation abrogates WRN exonuclease activity. (B) Schematic illustration of the eight WRN constructs utilized in the study. (C) DNA synthesis by hpol κ19-526 (2 nM) was monitored over time using a 13/18-mer primer-template DNA substrate (200 nM) in the absence of WRN and in the presence of full-length WRN1-1432 (100 nM). A schematic of the p/t-DNA substrate is shown above the gel results. (D) Total product formation [i.e. all of the product bands from panel (C)] was plotted as a function of time. The mean ± SEM is shown (n = 2). Product formation in each experiment was fit to a single-exponential equation [Equation (1)] to yield the following kinetic parameters: No WRN (black closed circles, •): A = 153 ± 4 nM, kobs = 0.099 ± 0.007 min−1; WRN1-1432 (blue closed squares, ): A = 171 ± 2 nM, kobs = 0.43 ± 0.01 min−1. (E) The relative activity of hpol κ in the presence of different WRN constructs is shown. Polymerase extension assays with additional WRN constructs were performed as described in panel (C). The rate constants for the total product formed were 0.43 ± 0.02 (hpol κ + WRN1-1092/E84A), 0.16 ± 0.01 (hpol κ + WRN1-949/E84A), 0.13 ± 0.01 (hpol κ + WRN500-1150), 0.18 ± 0.01 (hpol κ + WRN500-1092), 0.094 ± 0.008 (hpol κ + WRN500-949), 0.18 ± 0.01 (hpol κ + WRN949-1092) and 0.19 ± 0.01 (hpol κ + WRN1-333) nM min−1. The values reported represent the mean ± SEM (n = 2). The relative activity of hpol κ was calculated by dividing the rate constant for primer extension in the presence of the WRN construct by the rate constant for primer extension by hpol κ alone then multiplying by 100.
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Figure 1: WRN stimulates hpol κ19-526 activity on undamaged DNA templates. (A) An overview of the full-length WRN protein showing domains with either structural or catalytic properties relevant to the current study. HRDC: Helicase and RNaseD C-terminal; NLS: nuclear localization signal; PIP: PCNA interacting peptide; RQC: RecQ C-terminal; the E84A mutation abrogates WRN exonuclease activity. (B) Schematic illustration of the eight WRN constructs utilized in the study. (C) DNA synthesis by hpol κ19-526 (2 nM) was monitored over time using a 13/18-mer primer-template DNA substrate (200 nM) in the absence of WRN and in the presence of full-length WRN1-1432 (100 nM). A schematic of the p/t-DNA substrate is shown above the gel results. (D) Total product formation [i.e. all of the product bands from panel (C)] was plotted as a function of time. The mean ± SEM is shown (n = 2). Product formation in each experiment was fit to a single-exponential equation [Equation (1)] to yield the following kinetic parameters: No WRN (black closed circles, •): A = 153 ± 4 nM, kobs = 0.099 ± 0.007 min−1; WRN1-1432 (blue closed squares, ): A = 171 ± 2 nM, kobs = 0.43 ± 0.01 min−1. (E) The relative activity of hpol κ in the presence of different WRN constructs is shown. Polymerase extension assays with additional WRN constructs were performed as described in panel (C). The rate constants for the total product formed were 0.43 ± 0.02 (hpol κ + WRN1-1092/E84A), 0.16 ± 0.01 (hpol κ + WRN1-949/E84A), 0.13 ± 0.01 (hpol κ + WRN500-1150), 0.18 ± 0.01 (hpol κ + WRN500-1092), 0.094 ± 0.008 (hpol κ + WRN500-949), 0.18 ± 0.01 (hpol κ + WRN949-1092) and 0.19 ± 0.01 (hpol κ + WRN1-333) nM min−1. The values reported represent the mean ± SEM (n = 2). The relative activity of hpol κ was calculated by dividing the rate constant for primer extension in the presence of the WRN construct by the rate constant for primer extension by hpol κ alone then multiplying by 100.

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.


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 hpol κ19-526 activity on undamaged DNA templates. (A) An overview of the full-length WRN protein showing domains with either structural or catalytic properties relevant to the current study. HRDC: Helicase and RNaseD C-terminal; NLS: nuclear localization signal; PIP: PCNA interacting peptide; RQC: RecQ C-terminal; the E84A mutation abrogates WRN exonuclease activity. (B) Schematic illustration of the eight WRN constructs utilized in the study. (C) DNA synthesis by hpol κ19-526 (2 nM) was monitored over time using a 13/18-mer primer-template DNA substrate (200 nM) in the absence of WRN and in the presence of full-length WRN1-1432 (100 nM). A schematic of the p/t-DNA substrate is shown above the gel results. (D) Total product formation [i.e. all of the product bands from panel (C)] was plotted as a function of time. The mean ± SEM is shown (n = 2). Product formation in each experiment was fit to a single-exponential equation [Equation (1)] to yield the following kinetic parameters: No WRN (black closed circles, •): A = 153 ± 4 nM, kobs = 0.099 ± 0.007 min−1; WRN1-1432 (blue closed squares, ): A = 171 ± 2 nM, kobs = 0.43 ± 0.01 min−1. (E) The relative activity of hpol κ in the presence of different WRN constructs is shown. Polymerase extension assays with additional WRN constructs were performed as described in panel (C). The rate constants for the total product formed were 0.43 ± 0.02 (hpol κ + WRN1-1092/E84A), 0.16 ± 0.01 (hpol κ + WRN1-949/E84A), 0.13 ± 0.01 (hpol κ + WRN500-1150), 0.18 ± 0.01 (hpol κ + WRN500-1092), 0.094 ± 0.008 (hpol κ + WRN500-949), 0.18 ± 0.01 (hpol κ + WRN949-1092) and 0.19 ± 0.01 (hpol κ + WRN1-333) nM min−1. The values reported represent the mean ± SEM (n = 2). The relative activity of hpol κ was calculated by dividing the rate constant for primer extension in the presence of the WRN construct by the rate constant for primer extension by hpol κ alone then multiplying by 100.
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Related In: Results  -  Collection

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Figure 1: WRN stimulates hpol κ19-526 activity on undamaged DNA templates. (A) An overview of the full-length WRN protein showing domains with either structural or catalytic properties relevant to the current study. HRDC: Helicase and RNaseD C-terminal; NLS: nuclear localization signal; PIP: PCNA interacting peptide; RQC: RecQ C-terminal; the E84A mutation abrogates WRN exonuclease activity. (B) Schematic illustration of the eight WRN constructs utilized in the study. (C) DNA synthesis by hpol κ19-526 (2 nM) was monitored over time using a 13/18-mer primer-template DNA substrate (200 nM) in the absence of WRN and in the presence of full-length WRN1-1432 (100 nM). A schematic of the p/t-DNA substrate is shown above the gel results. (D) Total product formation [i.e. all of the product bands from panel (C)] was plotted as a function of time. The mean ± SEM is shown (n = 2). Product formation in each experiment was fit to a single-exponential equation [Equation (1)] to yield the following kinetic parameters: No WRN (black closed circles, •): A = 153 ± 4 nM, kobs = 0.099 ± 0.007 min−1; WRN1-1432 (blue closed squares, ): A = 171 ± 2 nM, kobs = 0.43 ± 0.01 min−1. (E) The relative activity of hpol κ in the presence of different WRN constructs is shown. Polymerase extension assays with additional WRN constructs were performed as described in panel (C). The rate constants for the total product formed were 0.43 ± 0.02 (hpol κ + WRN1-1092/E84A), 0.16 ± 0.01 (hpol κ + WRN1-949/E84A), 0.13 ± 0.01 (hpol κ + WRN500-1150), 0.18 ± 0.01 (hpol κ + WRN500-1092), 0.094 ± 0.008 (hpol κ + WRN500-949), 0.18 ± 0.01 (hpol κ + WRN949-1092) and 0.19 ± 0.01 (hpol κ + WRN1-333) nM min−1. The values reported represent the mean ± SEM (n = 2). The relative activity of hpol κ was calculated by dividing the rate constant for primer extension in the presence of the WRN construct by the rate constant for primer extension by hpol κ alone then multiplying by 100.
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.

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