1. Volume 41, Issue 5, Pages 609-615 (March 2011)
USP47 Is a Deubiquitylating Enzyme that Regulates Base Excision Repair by Controlling Steady-State Levels of DNA Polymerase β 
Jason L. Parsons, Irina I. Dianova, Svetlana V. Khoronenkova, Mariola J. Edelmann, Benedikt M. Kessler, Grigory L. Dianov 
Molecular Cell 
Volume 41, Issue 5, Pages (March 2011)
DOI: /j.molcel
Copyright © 2011 Elsevier Inc. Terms and Conditions

2. Molecular Cell 2011 41, 609-615DOI: (10.1016/j.molcel.2011.02.016)
Copyright © 2011 Elsevier Inc. Terms and Conditions

3. Figure 1 Purification of the Deubiquitylation Activity for Pol β
(A) His-tagged Pol β (5 pmol) was ubiquitylated by Mule partially purified from HeLa whole-cell extracts in the presence of E1 (0.7 pmol), H7 E2 (9.5 pmol), and ubiquitin (0.6 nmol). Ubiquitylated Pol β was purified from the reaction by Ni-NTA magnetic agarose beads. M stands for molecular weight markers.
(B) In vitro deubiquitylation of ubiquitylated Pol β (2.5 pmol) by cytoplasmic (C) and nuclear (N) fractions (1 μg) isolated from HeLa cells. NP refers to a control reaction incubated in the absence of a protein fraction.
(C) Purification scheme for the deubiquitylation enzyme for Pol β from HeLa cells.
(D) In vitro deubiquitylation of ubiquitylated Pol β (2.5 pmol) by final Mini Q chromatography fractions purified from HeLa cells.
(E) The peak active fraction (indicated with a vertical arrow in Figure 1D) was used for pull-down by the deubiquitylation enzyme-specific probe HA-Ub-Br2, and proteins were silver stained. Bands were analyzed by tandem mass spectrometry and were shown to contain the deubiquitylation enzymes USP5, USP15, and USP47. Where appropriate, proteins were analyzed by 10% SDS-PAGE and immunoblotted using the indicated antibodies; molecular weight markers are indicated on the left hand side of the figures, and the positions of ubiquitylated Pol β (Pol βub) are shown.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 2 USP47 Is the Deubiquitylation Enzyme for Pol β
(A) In vitro deubiquitylation of ubiquitylated Pol β (2.5 pmol) by recombinant USP5 (0.5–2 pmol) and USP15 (0.5–2 pmol) was performed at 30°C for 60 min. (B) Active fraction from final Mini Q chromatography column purified from HeLa cells was mock or immunodepleted using USP15 or USP47-specific antibodies. Proteins bound to the beads were subsequently analyzed by 10% SDS-PAGE and immunoblotting using USP15 or USP47 antibodies or (C) used in an in vitro deubiquitylation assay against ubiquitylated Pol β. UT refers to a control reaction incubated in the absence of an immunoprecipitate. (D) USP47 full-length cDNA was used in an in vitro wheat germ cell-free expression system to generate full-length his-tagged protein and analyzed (1 μl of 50 μl reaction) by 10% SDS-PAGE and immunoblotting using USP47 antibodies. (E) Cell-free expression reactions performed in the absence (Mock) and presence of USP47 cDNA were incubated with Ni-NTA magnetic agarose beads to bind his-tagged USP47 protein, and the beads used in an in vitro deubiquitylation assay against ubiquitylated Pol β. Control reactions did not contain any beads. (F) HeLa cells were treated with Lipofectamine transfection reagent (10 μl) in the absence and presence of a mammalian expression plasmid encoding usp47 cDNA (4 μg) for 24 hr. Whole-cell extracts were prepared and analyzed by 10% SDS-PAGE and immunoblotting using USP47 antibodies. (G) His-tagged USP47 expressed in HeLa cells was purified using Ni-NTA agarose (Mock PD refers to pull-down from cells treated with Lipofectamine only) and analyzed by 10% SDS-PAGE and immunoblotting using USP47 antibodies or analyzed for in vitro deubiquitylation activity against monoubiquitylated Pol β (H, Pol βub) or polyubiquitylated Pol β (I, Pol βpolyub). Molecular weight markers are indicated on the left hand side of appropriate figures.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 3 USP47 Modulates the Cellular Level of Pol β
(A) HeLa cells were treated with Lipofectamine transfection reagent (10 μl) in the absence and presence of USP47 siRNA (200 pmol) for 72 hr, and whole-cell extracts were prepared.
(B) HeLa cells were treated with Lipofectamine transfection reagent (10 μl) in the presence and absence of USP47 siRNA (200 pmol) for 72 hr. Cytoplasmic (C) and nuclear (N) fractions were prepared and 40 μg protein in the C fraction and an equal volume of the N fraction were analyzed. Cytoplasmic levels of Pol β were 2.1 ± 0.9-fold (p < ) lower and Pol βub were 1.5 ± 0.3-fold (p < 0.02) higher in USP47 knockdown cells compared to Lipofectamine-only treated cells, analyzed by Student's t test from five independent experiments.
(C) Cytoplasmic (C) and nuclear (N) fractions were prepared from HeLa cells, and 40 μg protein in the C fraction and an equal volume of the N fraction were analyzed.
(D) HeLa cells were treated with Lipofectamine transfection reagent (10 μl) in the absence and presence of USP15 or USP47 siRNA (200 pmol) for 72 hr, and whole-cell extracts were prepared.
(E and F) HeLa cells were treated with Lipofectamine transfection reagent (10 μl) in the absence and presence of USP15, USP47, and/or Mule siRNA (200 pmol) for 48 hr. Cells were then treated with Lipofectamine transfection reagent (10 μl) in the absence and presence of a Flag-Pol β mammalian expression plasmid (300 fmol) for a further 24 hr, and whole-cell extracts were prepared. Flag-Pol β levels were 3.1 ± 1.0-fold lower in USP47 knockdown cells compared to Lipofectamine-only treated cells, p < by Student's t test from four independent experiments. Where appropriate, proteins were analyzed by 10% SDS-PAGE and immunoblotted using the indicated antibodies.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 4 USP47 Modulates Base Excision Repair
HeLa cells were treated with Lipofectamine (10 μl) in the absence and presence of USP47 shRNA (1 μg) for 48 hr. Whole-cell extracts were prepared and analyzed by 10% SDS-PAGE and immunoblotting with antibodies directed against (A) poly(ADP-ribose) polymers or (B) ARF, in comparison to actin as a loading control. ARF protein levels were 1.3 ± 0.1-fold higher in USP47 knockdown cells compared to Lipofectamine-only treated cells, p < 0.02 by Student's t test from three independent experiments. (C and D) AG06173 primary fibroblasts were treated with Lipofectamine (10 μl) in the absence and presence of USP47 siRNA (200 pmol) for 72 hr. Cells were treated with 10 mM hydrogen peroxide for 15 min, and cytoplasmic (C) and nuclear (D) fractions were prepared and 40 μg protein were analyzed by 10% SDS-PAGE and immunoblotting with the antibodies indicated. Pol β cytoplasmic and nuclear protein levels were 1.5 ± 0.3-fold (p < 0.01) and 1.8 ± 0.4-fold (p < 0.005) lower, respectively, in USP47 knockdown cells compared to Lipofectamine-only treated cells. Furthermore, the levels of nuclear Pol β in Lipofectamine-only treated cells increased 1.2 ± 0.02-fold (p < ) following hydrogen peroxide treatment, analyzed by Student's t test from three independent experiments. (E and F) HeLa cells were treated with Lipofectamine (10 μl) in the absence and presence of USP15 or USP47 siRNA (200 pmol) for 72 hr. The cells were then treated with (E) 20 μM hydrogen peroxide for 5 min or (F) 1.5 mM MMS for 30 min, allowed to repair for 0–120 min, and the levels of SSBs and alkali labile sites then analyzed by the alkaline single-cell gel electrophoresis (Comet) assay. Shown are the mean percent tail DNA values with standard deviations from at least three independent experiments. Statistically significant results comparing Lipofectamine and USP47 siRNA-treated cells are represented by ∗p < 0.01 and ∗∗p < 0.001, as analyzed by Student's t test. (G) HeLa cells were treated with Lipofectamine (10 μl) in the absence and presence of USP47 shRNA (1 μg) for 48 hr and clonogenic survival measured following treatment of the cells with hydrogen peroxide. Shown are the mean percent survival values with standard deviations from at least three independent experiments. (H) Proposed model for the regulation of BER by Mule, CHIP, and USP47. Newly synthesized Pol β is either directly transferred to the nucleus (1) or ubiquitylated by Mule (2). Ubiquitylated Pol β is then a target for CHIP-mediated polyubiquitylation (3) and subsequent degradation by the proteasome (4). However, if DNA damage is detected, the activity of Mule is inhibited by ARF (5) and USP47 is then able to deubiquitylate mono- or polyubiquitylated Pol β (6), which then generates more active Pol β that is able to enter the nucleus to participate in DNA repair.
Molecular Cell  , DOI: ( /j.molcel )
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