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Human DNA polymerase beta polymorphism, Arg137Gln, impairs its polymerase activity and interaction with PCNA and the cellular base excision repair capacity.

Guo Z, Zheng L, Dai H, Zhou M, Xu H, Shen B - Nucleic Acids Res. (2009)

Bottom Line: More than 30% of human tumors characterized to date express DNA Pol beta variants, many of which result from a single nucleotide residue substitution.The substitution also affects the interaction between Pol beta and proliferating cell nuclear antigen (PCNA).Expression of wild-type Pol beta in pol beta(-/-) mouse embryonic fibroblast (MEF) cells restored cellular resistance to DNA damaging reagents such as methyl methanesulfonate (MMS) and N-methyl-N-nitrosourea (MNU), while expression of R137Q in pol beta(-/-) MEF cells failed to do so.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiation Biology, City of Hope National Medical Center, Beckman Research Institute, Duarte, CA 91010, USA.

ABSTRACT
DNA polymerase beta (Pol beta) is a key enzyme in DNA base excision repair, and an important factor for maintaining genome integrity and stability. More than 30% of human tumors characterized to date express DNA Pol beta variants, many of which result from a single nucleotide residue substitution. However, in most cases, their precise functional deficiency and relationship to cancer susceptibility are still unknown. In the current work, we show that a polymorphism encoding an arginine to glutamine substitution, R137Q, has lower polymerase activity. The substitution also affects the interaction between Pol beta and proliferating cell nuclear antigen (PCNA). These defects impair the DNA repair capacity of Pol beta in reconstitution assays, as well as in cellular extracts. Expression of wild-type Pol beta in pol beta(-/-) mouse embryonic fibroblast (MEF) cells restored cellular resistance to DNA damaging reagents such as methyl methanesulfonate (MMS) and N-methyl-N-nitrosourea (MNU), while expression of R137Q in pol beta(-/-) MEF cells failed to do so. These data indicate that polymorphisms in base excision repair genes may contribute to the onset and development of cancers.

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The Pol β polymorphism R137Q is defective in polymerase activity. (A) Top panel, schematic of the biotin-labeled 1-nt gapped DNA substrate (Pol-GAP); bottom panel, polymerase activity assay in which Pol-GAP was incubated with 50 μM each of dATP, dGTP, dTTP and 8 μM dCTP-32P and varying amounts of purified Pol β protein (WT and R137Q). WT and R137Q DNA polymerization products were separated by denaturing gel electrophoresis and visualized with a phosphorimager. (B) DNA polymerization products were pulled down by Sepharose–avidin beads. After washing, the amount of radio-nucleotide incorporated into the products was determined by liquid scintillation counting. (C) Gel shift assay of DNA-binding affinity of R137Q and WT Pol β. In this assay, Pol-GAP was labeled by 32P as shown in figure. (D) ELISA-based isotherm adsorption assay of DNA-binding affinity of R137Q and WT Pol β. The DNA substrate was the same as that used in (A). WT, filled squares; R137, filled circles.
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Figure 1: The Pol β polymorphism R137Q is defective in polymerase activity. (A) Top panel, schematic of the biotin-labeled 1-nt gapped DNA substrate (Pol-GAP); bottom panel, polymerase activity assay in which Pol-GAP was incubated with 50 μM each of dATP, dGTP, dTTP and 8 μM dCTP-32P and varying amounts of purified Pol β protein (WT and R137Q). WT and R137Q DNA polymerization products were separated by denaturing gel electrophoresis and visualized with a phosphorimager. (B) DNA polymerization products were pulled down by Sepharose–avidin beads. After washing, the amount of radio-nucleotide incorporated into the products was determined by liquid scintillation counting. (C) Gel shift assay of DNA-binding affinity of R137Q and WT Pol β. In this assay, Pol-GAP was labeled by 32P as shown in figure. (D) ELISA-based isotherm adsorption assay of DNA-binding affinity of R137Q and WT Pol β. The DNA substrate was the same as that used in (A). WT, filled squares; R137, filled circles.

Mentions: The polymerase activity assay utilized 50 mM Tris–HCl (pH 8.0), 10 mM MgCl2, 2 mM DTT, 20 mM NaCl, 10% glycerol, 0.1 µM biotin-labeled DNA substrate Pol-GAP (see Table 1 and Figure 1A for detail), 50 µM each dATP, dGTP and dTTP (Sigma), 8 µM 2 µCi [α-32P]-dCTP and 0–10 ng WT or R137Q Pol β. Reactions were carried out at 37°C for 30 min. A portion of reaction product was taken out and incubated with avidin–Sepharose 4B beads, washed and quantified on liquid scintillation analyzer. The left portion of reaction product was then stopped by addition of equal volumes of gel loading buffer (90% formamide dye, 3 M EDTA, 0.02% bromophenol blue and 0.02% xylene cyanol), heated (5 min, 95°C), separated by 15% Polyacrylamide gel electrophoresis (PAGE) containing 8 M urea and visualized by autoradiography.Figure 1.


Human DNA polymerase beta polymorphism, Arg137Gln, impairs its polymerase activity and interaction with PCNA and the cellular base excision repair capacity.

Guo Z, Zheng L, Dai H, Zhou M, Xu H, Shen B - Nucleic Acids Res. (2009)

The Pol β polymorphism R137Q is defective in polymerase activity. (A) Top panel, schematic of the biotin-labeled 1-nt gapped DNA substrate (Pol-GAP); bottom panel, polymerase activity assay in which Pol-GAP was incubated with 50 μM each of dATP, dGTP, dTTP and 8 μM dCTP-32P and varying amounts of purified Pol β protein (WT and R137Q). WT and R137Q DNA polymerization products were separated by denaturing gel electrophoresis and visualized with a phosphorimager. (B) DNA polymerization products were pulled down by Sepharose–avidin beads. After washing, the amount of radio-nucleotide incorporated into the products was determined by liquid scintillation counting. (C) Gel shift assay of DNA-binding affinity of R137Q and WT Pol β. In this assay, Pol-GAP was labeled by 32P as shown in figure. (D) ELISA-based isotherm adsorption assay of DNA-binding affinity of R137Q and WT Pol β. The DNA substrate was the same as that used in (A). WT, filled squares; R137, filled circles.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2691839&req=5

Figure 1: The Pol β polymorphism R137Q is defective in polymerase activity. (A) Top panel, schematic of the biotin-labeled 1-nt gapped DNA substrate (Pol-GAP); bottom panel, polymerase activity assay in which Pol-GAP was incubated with 50 μM each of dATP, dGTP, dTTP and 8 μM dCTP-32P and varying amounts of purified Pol β protein (WT and R137Q). WT and R137Q DNA polymerization products were separated by denaturing gel electrophoresis and visualized with a phosphorimager. (B) DNA polymerization products were pulled down by Sepharose–avidin beads. After washing, the amount of radio-nucleotide incorporated into the products was determined by liquid scintillation counting. (C) Gel shift assay of DNA-binding affinity of R137Q and WT Pol β. In this assay, Pol-GAP was labeled by 32P as shown in figure. (D) ELISA-based isotherm adsorption assay of DNA-binding affinity of R137Q and WT Pol β. The DNA substrate was the same as that used in (A). WT, filled squares; R137, filled circles.
Mentions: The polymerase activity assay utilized 50 mM Tris–HCl (pH 8.0), 10 mM MgCl2, 2 mM DTT, 20 mM NaCl, 10% glycerol, 0.1 µM biotin-labeled DNA substrate Pol-GAP (see Table 1 and Figure 1A for detail), 50 µM each dATP, dGTP and dTTP (Sigma), 8 µM 2 µCi [α-32P]-dCTP and 0–10 ng WT or R137Q Pol β. Reactions were carried out at 37°C for 30 min. A portion of reaction product was taken out and incubated with avidin–Sepharose 4B beads, washed and quantified on liquid scintillation analyzer. The left portion of reaction product was then stopped by addition of equal volumes of gel loading buffer (90% formamide dye, 3 M EDTA, 0.02% bromophenol blue and 0.02% xylene cyanol), heated (5 min, 95°C), separated by 15% Polyacrylamide gel electrophoresis (PAGE) containing 8 M urea and visualized by autoradiography.Figure 1.

Bottom Line: More than 30% of human tumors characterized to date express DNA Pol beta variants, many of which result from a single nucleotide residue substitution.The substitution also affects the interaction between Pol beta and proliferating cell nuclear antigen (PCNA).Expression of wild-type Pol beta in pol beta(-/-) mouse embryonic fibroblast (MEF) cells restored cellular resistance to DNA damaging reagents such as methyl methanesulfonate (MMS) and N-methyl-N-nitrosourea (MNU), while expression of R137Q in pol beta(-/-) MEF cells failed to do so.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiation Biology, City of Hope National Medical Center, Beckman Research Institute, Duarte, CA 91010, USA.

ABSTRACT
DNA polymerase beta (Pol beta) is a key enzyme in DNA base excision repair, and an important factor for maintaining genome integrity and stability. More than 30% of human tumors characterized to date express DNA Pol beta variants, many of which result from a single nucleotide residue substitution. However, in most cases, their precise functional deficiency and relationship to cancer susceptibility are still unknown. In the current work, we show that a polymorphism encoding an arginine to glutamine substitution, R137Q, has lower polymerase activity. The substitution also affects the interaction between Pol beta and proliferating cell nuclear antigen (PCNA). These defects impair the DNA repair capacity of Pol beta in reconstitution assays, as well as in cellular extracts. Expression of wild-type Pol beta in pol beta(-/-) mouse embryonic fibroblast (MEF) cells restored cellular resistance to DNA damaging reagents such as methyl methanesulfonate (MMS) and N-methyl-N-nitrosourea (MNU), while expression of R137Q in pol beta(-/-) MEF cells failed to do so. These data indicate that polymorphisms in base excision repair genes may contribute to the onset and development of cancers.

Show MeSH
Related in: MedlinePlus