Journal: Nucleic Acids Research

Article Title: Human DNA polymerase β polymorphism, Arg137Gln, impairs its polymerase activity and interaction with PCNA and the cellular base excision repair capacity

doi: 10.1093/nar/gkp201

Figure Lengend Snippet: 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- 32 P 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 32 P 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.

Article Snippet: Bound Pol β was detected by mouse anti-Pol β antibody (Genetex, GTX23181) and goat anti-mouse IgG/HRP (Genetex, GTX85313).

Techniques: Activity Assay, Labeling, Incubation, Purification, Nucleic Acid Electrophoresis, Avidin-Biotin Assay, Gel Shift, Binding Assay, Enzyme-linked Immunosorbent Assay, Adsorption

Journal: Nucleic Acids Research

Article Title: Human DNA polymerase β polymorphism, Arg137Gln, impairs its polymerase activity and interaction with PCNA and the cellular base excision repair capacity

doi: 10.1093/nar/gkp201

Figure Lengend Snippet: dRP lyase activity assay of Pol β. ( A ) Schematic representation of the reactions generating a 5′-dRP-group-containing DNA substrate. Treatment of Pol β-U (with 32 P labeled at 3′end) with UDG generates an AP-containing DNA, which then was incised at the nucleotide 5′ to the AP site by APE1, resulting the 5′-dRP-containing substrate (20 nt + dRP). Removal of the 5′-dRP residue by dRP lyase results in a 3′- 32 P labeled 20 nt DNA fragment. Asterisk represents 32 P labeling. ( B ) 5′-dRP lyase activity assay. ( C ) Quantification of the 5′-dRP lyase activities shown in (B).

Article Snippet: Bound Pol β was detected by mouse anti-Pol β antibody (Genetex, GTX23181) and goat anti-mouse IgG/HRP (Genetex, GTX85313).

Techniques: Activity Assay, Labeling, Residue

Journal: Nucleic Acids Research

Article Title: Human DNA polymerase β polymorphism, Arg137Gln, impairs its polymerase activity and interaction with PCNA and the cellular base excision repair capacity

doi: 10.1093/nar/gkp201

Figure Lengend Snippet: The R137Q Pol β variant has an impaired interaction with PCNA. ( A ) Ni-NTA pull-down was performed with the cell extracts from pol β null MEFs and purified Pol β or R137Q variant proteins (His-tagged). The interaction was detected by western blotting using antibodies against FEN1, APE1, PCNA, Ligase 1, Ligase IIIα and XRCC1. A negative control with His-tagged GST was added to rule out the possibility that desirable proteins interact with His tag directly. ( B ) Pull-down assay using purified proteins. Purified PCNA was immobilized on Sepharose 4B beads, incubated with purified Pol β (WT or R137Q), washed and beads were then boiled in SDS–PAGE sample buffer. Proteins were separated by SDS–PAGE and transferred onto PVDF film, followed by detection with anti-Pol β and anti-PCNA antibodies. ( C ) Affinity comparison of PCNA with WT and R137Q Pol β by ELISA. PCNA was coated onto ELISA plates, incubated with different concentrations of purified WT or R137Q Pol β, washed and bound protein was detected by mouse anti-Pol β antibody and goat anti-mouse IgG/HRP. Color was developed by adding TMB. The color reaction was stopped by adding 1 N HCl. Optical density was read on microplate reader.

Article Snippet: Bound Pol β was detected by mouse anti-Pol β antibody (Genetex, GTX23181) and goat anti-mouse IgG/HRP (Genetex, GTX85313).

Techniques: Variant Assay, Purification, Western Blot, Negative Control, Pull Down Assay, Incubation, SDS Page, Comparison, Enzyme-linked Immunosorbent Assay

Journal: Nucleic Acids Research

Article Title: Human DNA polymerase β polymorphism, Arg137Gln, impairs its polymerase activity and interaction with PCNA and the cellular base excision repair capacity

doi: 10.1093/nar/gkp201

Figure Lengend Snippet: R137Q significantly reduces BER efficiency. ( A ) SP-BER reconstitution with purified WT and R137Q Pol β. ( B ) LP-BER reconstitution with purified WT and R137Q. The top part of each panel shows the schematic structures of the corresponding DNA substrates. The middle shows PAGE-separated products and the bottom the relative percentage of repaired product obtained with the indicated amounts of Pol β. Values represent mean ± SD of three independent assays. WT, filled squares; R137, filled circle.

Article Snippet: Bound Pol β was detected by mouse anti-Pol β antibody (Genetex, GTX23181) and goat anti-mouse IgG/HRP (Genetex, GTX85313).

Techniques: Purification

Journal: Nucleic Acids Research

Article Title: Human DNA polymerase β polymorphism, Arg137Gln, impairs its polymerase activity and interaction with PCNA and the cellular base excision repair capacity

doi: 10.1093/nar/gkp201

Figure Lengend Snippet: Polymorphic Pol β R137Q is defective in BER reconstitution using whole cell extract. ( A ) Western blotting shows the establishment of cell lines that express the Pol β WT protein or polymorphic Pol β R137Q in pol β −/− MEF cells. Amount of Pol β protein was detected in 100 µg of total cell extract. ( B ) SP-BER reconstitution with WT and R137Q. ( C ) LP-BER reconstitution with WT and R137Q. The top part of each panel shows the schematic structure of the corresponding DNA substrates. The middle shows PAGE-separated products and the bottom the relative percentage of repaired product at different enzyme concentrations, as indicated. Values represent mean ± SD of three independent assays. WT, filled squares; pol β −/− /WT, filled triangles; pol β −/− , filled circles; pol β −/− /R137Q, open squares.

Article Snippet: Bound Pol β was detected by mouse anti-Pol β antibody (Genetex, GTX23181) and goat anti-mouse IgG/HRP (Genetex, GTX85313).

Techniques: Western Blot

Journal: Nucleic Acids Research

Article Title: Human DNA polymerase β polymorphism, Arg137Gln, impairs its polymerase activity and interaction with PCNA and the cellular base excision repair capacity

doi: 10.1093/nar/gkp201

Figure Lengend Snippet: Cells harboring polymorphism R137Q are sensitive to DNA damage stress. ( A ) Abasic-specific DNA damage assay. Cells were treated with MMS. The amount of damaged DNA lesions was then detected by ARP reagent (see Experimental Procedures section). ( B ) Effect of MMS on induction of apoptosis in complemented pol β −/− MEFs. Pol β −/− MEFs complemented with WT or the R137Q variant of Pol β were treated (1 h) with 2 mM MMS. Adherent and floating cells were harvested after 24 h and analyzed by flow cytometry. The percentage of the apoptotic cells indicated. The MMS-induced increase of apoptotic cells is shown in the bottom bar. WT, pol β −/− , pol β −/− /WT and pol β −/− /R137Q MEF cells were treated (1 h) with MMS ( C ) and MNU ( D ) at the indicated concentrations. Cellular sensitivity was determined by growth inhibition experiments. Data represents mean ± SD of four independent experiments. WT, filled squares; pol β −/− /WT, filled triangles; pol β −/− , filled circles; pol β −/− /R137Q, open squares.

Article Snippet: Bound Pol β was detected by mouse anti-Pol β antibody (Genetex, GTX23181) and goat anti-mouse IgG/HRP (Genetex, GTX85313).

Techniques: Variant Assay, Flow Cytometry, Inhibition