1. Volume 29, Issue 4, Pages 477-487 (February 2008)
CHIP-Mediated Degradation and DNA Damage-Dependent Stabilization Regulate Base Excision Repair Proteins 
Jason L. Parsons, Phillip S. Tait, David Finch, Irina I. Dianova, Sarah L. Allinson, Grigory L. Dianov 
Molecular Cell 
Volume 29, Issue 4, Pages (February 2008)
DOI: /j.molcel
Copyright © 2008 Elsevier Inc. Terms and Conditions

2. Figure 1 Reduced Level of Pol β and Lig III in XRCC1-Deficient Cells
(A and C) Whole-cell extracts were prepared from the indicated cells, and proteins were analyzed by western blotting with the indicated antibodies. Note that the human XRCC1 antibody does not crossreact with hamster XRCC1 protein (shown as undetectable, UD).
(B) XRCC1-deficent cells (EM-C11) and EM-C11 cells transfected with plasmid expressing human XRCC1 protein (EM-C11-XH) were fixed and immunostained with the indicated polyclonal antibodies.
(D and E) XRCC1-deficient cells transfected with empty vector (EM9-V) and EM9 cells transfected with plasmid expressing human XRCC1 protein (EM9-XH) were treated with cycloheximide (50 μg/ml) for the times indicated. Whole-cell extracts were prepared and analyzed by immunoblotting with the indicated antibodies.
(F) Whole-cell extracts were prepared from untreated HeLa cells (U), cells treated with scrambled oligonucleotide for 72 hr (S), and cells treated with XRCC1-silencing oligonucleotide for 24, 48, and 72 hr. Proteins were analyzed by western blotting with the indicated antibodies.
(G) Cell extracts shown in Figure 1F were also probed with antibodies raised against poly(ADP-ribose) (PAR) polymers.
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2
XRCC1-Dependent Binding of Pol β to Chromatin in Response to DNA Damage
(A–C) Whole-cell extracts from untreated and hydrogen peroxide (10 mM for 15 min) treated XRCC1-deficient cells transfected with plasmid expressing human XRCC1 protein (EM-C11-XH) were prepared and his-tagged XRCC1 purified with Ni-agarose beads. Proteins in the extracts (C) and in the pull down (A) were analyzed by western blotting with the corresponding antibodies. (B) Graphical representation of hydrogen peroxide stimulation of Pol β-XRCC1 interaction observed in (A) normalized to the amount of XRCC1, where Pol β levels in untreated cells are set to 1 and the levels observed after hydrogen peroxide treatment include standard deviations from at least three independent experiments. Untreated cells (D) and cells treated with hydrogen peroxide (E) were lysed and subjected to fractionation into cytoplasmic (C) and chromatin-bound (CB) fractions. Proteins were analyzed by western blotting with Pol β antibodies.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3
Purification of E3 Ubiquitin Ligase Activity for BER Proteins from HeLa Whole-Cell Extracts
(A) Purification scheme for the isolation of E3 ubiquitin ligase from HeLa cell extracts. (B) E3 ubiquitin ligase activity of the final Mono Q fractions (B1-C4) against Pol β protein detected by western blotting and (C) comparison of activity of Fraction C2 against Pol β protein in the presence of normal (Wt) or mutant (Mut) ubiquitin that is unable to form polyubiquitylation chains. Specificity of ubiquitin ligase activity of Fraction C2 against Pol β (D) and XRCC1 (E) proteins demonstrating the requirement for both fraction, containing E3 ubiquitin ligase activity, and ubiquitin. Molecular weight markers are indicated on the left-hand side of the appropriate figures.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4
Identification of CHIP as an E3 Ubiquitin Ligase Active against BER Proteins
(A) Amino acid sequence of CHIP, and highlighted in bold are the peptides identified by mass spectrometry analysis of Fraction C2 from the final Mono Q column from purification of HeLa whole-cell extracts.
(B) Analysis of recombinant his-tagged CHIP by the Experion Electrophoresis System after purification by Ni-NTA agarose and Mono Q chromatography.
(C) In vitro ubiquitylation of BER proteins (5 pmol) by CHIP (14 pmol) were analyzed by western blot analysis using the corresponding antibodies.
(D) In vitro ubiquitylation of Pol β by wild-type (WT) and H260Q U box mutant of CHIP that is unable to interact with the E2 conjugating enzyme.
(E) Degradation of CHIP ubiquitylated Pol β by the 26S proteasome in vitro.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 5 CHIP Targets BER Proteins for Degradation in Human Cells
(A–C) HeLa cells were transiently cotransfected with plasmids expressing FLAG-tagged CHIP, his-tagged XRCC1, his-tagged Pol β, or empty vector. Whole-cell extracts were prepared, and protein levels were analyzed by western blotting using Pol β antibodies (reveals the level of endogenous and transfected Pol β), FLAG antibodies (for FLAG-CHIP), his-tagged antibodies (for His-XRCC1), or actin antibodies as a loading control.
(D) HeLa cells were transfected with siRNA directed against CHIP for 72 hr, RNA was isolated, and the levels of CHIP mRNA were determined by RT-PCR analysis.
(E) HeLa cells were transfected with siRNA directed against CHIP for 72 hr, whole-cell extracts were prepared, and protein levels were analyzed by western blot analysis using the corresponding antibodies.
(F) HeLa cells were transiently cotransfected with plasmids expressing the K30A TPR mutant of CHIP (unable to interact with hsp70 and hsp90) and Pol β, whole-cell extracts were prepared, and protein levels were analyzed by western blotting using Pol β antibodies (reveals the level of endogenous and transfected Pol β), FLAG antibodies (for FLAG-CHIP), or actin antibodies as a loading control.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 6
Determination of the Site of Ubiquitylation of Pol β and XRCC1 and Demonstration of Increased Stability of Truncated Proteins
(A) Schematic representation of Pol β protein showing the 8 and 31 kDa domains that can be generated by limited trypsin hydrolysis. Fraction C2 from Mono Q purification of HeLa whole-cell extracts was used to demonstrate in vitro ubiquitin ligase activity against 8 kDa (B), but not the 31 kDa (C), domain of Pol β by using mutant ubiquitin that is unable to form polyubiquitylation chains.
(D) HeLa cells were either untreated (UT) or transiently transfected with plasmid expressing full-length human Pol β protein (WT) or Pol β-31 kDa domain (31K), and whole-cell extracts were prepared and analyzed by western blotting using Pol β antibodies. The levels of endogenous and transfected Pol β can be seen.
(E) Schematic representation of XRCC1 protein showing the various domains and truncations of XRCC1 generated.
(F and G) In vitro ubiquitylation of N-terminal domain (XRCC11–164) and C-terminal fragment (XRCC1461–633) of XRCC1, respectively, by Fraction C2 using mutant ubiquitin that is unable to form polyubiquitylation chains.
(H) XRCC1-deficient (EM-C11) cells were either untreated (UT) or transiently transfected with plasmid expressing his-tagged full-length human XRCC1 protein (XH) or C-terminal deletion of XRCC1 (XHΔ529–633), and whole-cell extracts were prepared and analyzed by western blotting using XRCC1 antibodies.
(I) Whole-cell extracts from XRCC1-deficient (EM-C11) cells stably transfected with empty plasmid (V) or with a plasmid expressing his-tagged full-length human XRCC1 protein (XH) or C-terminal deletion of XRCC1 (XHΔ529–633) were prepared and analyzed by western blotting using XRCC1 antibodies. Molecular weight markers are indicated on the left hand side of the appropriate figures.
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 7 Proposed Model for the Regulation of BER Protein Levels
BER protein levels maintained in the nucleus are dependent on the amount of unrepaired DNA lesions and based on the different stability of free and DNA damage-bound forms of the proteins. In unstressed cells, a proportion of Pol β is bound in a complex with XRCC1-Lig III on damaged DNA, and these proteins are thus less prone to degradation by proteolytic enzymes. The remainder are more rapidly degraded by CHIP-mediated ubiquitylation and proteasomal degradation and subsequently removed from the nucleus. In the event of increased DNA damage, the balance between the two forms changes, such that more BER proteins accumulate in the nucleus to repair the arising DNA damage.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2008 Elsevier Inc. Terms and Conditions