1. Volume 43, Issue 4, Pages 649-662 (August 2011)
The hMsh2-hMsh6 Complex Acts in Concert with Monoubiquitinated PCNA and Pol η in Response to Oxidative DNA Damage in Human Cells 
Anastasia Zlatanou, Emmanuelle Despras, Tirzah Braz-Petta, Imenne Boubakour-Azzouz, Caroline Pouvelle, Grant S. Stewart, Satoshi Nakajima, Akira Yasui, Alexander A. Ishchenko, Patricia L. Kannouche 
Molecular Cell 
Volume 43, Issue 4, Pages (August 2011)
DOI: /j.molcel
Copyright © 2011 Elsevier Inc. Terms and Conditions

2. Molecular Cell 2011 43, 649-662DOI: (10.1016/j.molcel.2011.06.023)
Copyright © 2011 Elsevier Inc. Terms and Conditions

3. Figure 1
Kinetics of PCNA Monoubiquitination after Treatment with Different Genotoxic Agents
(A and B) MRC5 cells were exposed to UVC, MMS, and γ-irradiation (A) or treated with different oxidizing agents (H2O2, KBrO3, or menadione) (B) and were harvested at indicated time points. Ubiquitinated forms of PCNA were detected by western blot.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2011 Elsevier Inc. Terms and Conditions

4. Figure 2
PCNA Is Monoubiquitinated on K164 in a Rad18-Dependent but S Phase-Independent Manner after Oxidative Stress
(A) Nocodazole-treated cells were exposed to H2O2 and lysed immediately after treatment. Total extracts were analyzed by western blot (left). FACS profile of nocodazole-treated cells is shown (right).
(B) Confluent normal primary cells (AS198) were treated with H2O2, then harvested at the indicated time points. Total extracts were analyzed by western blot (left). FACS analysis of confluent AS198 cells is shown (right).
(C) Cells transfected with RAD18 siRNAs were treated with H2O2 then lysed immediately or UVC irradiated and harvested 6 hr later. PCNA ubiquitination was assessed by western blot.
(D) Cells transfected with pHA-PCNA WT, pHA-PCNAK164R, or empty vector were treated with H2O2, then immediately harvested. HA-tagged proteins were immunoprecipitated, and HA-PCNA was detected by western blot. NT, nontransfected.
(E) MRC5 cells were exposed to H2O2, then incubated for the indicated times before lysis. Total extracts were then analyzed by western blot using indicated antibodies.
(F) MRC5 cells were transfected with either a nontarget (NT) siRNA or USP1 siRNAs. Cells were treated with H2O2 and lysed immediately or 4 hr later. Alternatively, cells were UV irradiated and lysed 6 hr later. PCNA ubiquitination was assessed by western blot.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2011 Elsevier Inc. Terms and Conditions

5. Figure 3
The Monoubiquitination of PCNA Is Independent of Functional BER but Is Dependent on Msh2/Msh6 Complex in Human Cells after Oxidative Stress
(A) Cells transfected with XRCC1 (left) or POLB (right) siRNA pools were treated with H2O2, then incubated for the indicated times before lysis. PCNA ubiquitination was assessed by western blot.
(B) MRC5 cells were pretreated with PAR inhibitor 4-AN, then exposed to H2O2.The cells were then either immediately lysed or further incubated for the indicated periods. Lysates were analyzed by western blot using indicated antibodies.
(C) Cells transfected with MSH2 siRNA were treated with H2O2, then harvested at the indicated times. Lysates were analyzed by western blot using indicated antibodies.
(D) Primary cells transfected with MSH2 siRNA were exposed to H2O2 when cells reached the confluency and were processed as in (C).
(E and F) Primary cells transfected with indicated siRNAs were treated with H2O2 (E) or UV irradiated (F) and processed as in (C).
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2011 Elsevier Inc. Terms and Conditions

6. Figure 4
Processing of the Oxidative DNA Damage by MMR and BER Pathways
(A) Strategy for the assay of excision and resynthesis in the gapped strand containing 5ohU and/or 8-oxoG, based on the removal of site-specific cytosine methylation. SmaI sensitivity identifies strand excision and resynthesis at the hemimethylated reporter site 6 or 7 nucleotides 5′ from the lesion/mismatch site, while HindIII sensitivity identifies the repair of oxidized or mismatched nucleotides inside the corresponding restriction site.
(B) Control MMR nick-directed assay using a T/G mismatch with WT or MSH2−/− MEF extracts.
(C–F) MMR/BER repair assay on circular (unnicked) duplex DNA molecule containing 5ohU/G (C), on gapped DNA plasmid containing 8-oxoG (D), on gapped plasmid containing 5ohU/G (E), and on gapped and unnicked plasmid containing tandem 8-oxoG-5ohU/C-G lesion (F) using WT or MSH2−/− MEF extracts. After repair assays, all circular DNA substrates were linearized by AlwNI treatment and processed as detailed in the Supplemental Experimental Procedures.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2011 Elsevier Inc. Terms and Conditions

7. Figure 5
Monoubiquitinated PCNA and Polη Are Bound to Chromatin and Form a Complex after Oxidative Damage in a MSH2-Dependent Manner
(A) MRC5 cells were transfected with unspecific siRNA (NT) or MSH2 siRNA (Msh2), then exposed to 200 μM H2O2 before lysis. Soluble proteins were extracted in CSK buffer containing increasing NaCl concentrations (50, 100, 200, and 400 mM), and insoluble fractions or whole cell extracts (WCE) were analyzed by western blotting using indicated antibodies.
(B) Exponentially growing cells were exposed to 200 μM H2O2, then incubated for indicated times prior to harvesting. Coimmunoprecipitation was performed on crosslinked insoluble fractions, then immunoprecipitates were analyzed by western blot using indicated antibodies.
(C) Confluent primary fibroblasts were treated with 600 μM H2O2, and coimmunoprecipitations were carried out as in (B).
(D) HCT116 (MLH1−/−) or LoVo (MSH2−/−) cells were treated with 200 μM H2O2, and coimmunoprecipitations were performed as in (B).
(E) Cells stably expressing Polη (MRC5-Cl14) were transfected with pHA-PCNA WT, pHA-PCNAK164R (KR), or empty pHA (HA) and treated with H2O2 24 hr later. Immunoprecipitation was carried out on crosslinked insoluble fractions, and proteins bound were analyzed by western blot using indicated antibodies. hc and lc, IgG chains; ∗, unspecific band.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2011 Elsevier Inc. Terms and Conditions

8. Figure 6
Accumulation of Polη at Sites of Oxidative DNA Damage Induced by UVA Laser Irradiation
(A) HeLa cells transiently expressing GFP-Polη were irradiated with laser light. GFP signals before (top) and after (bottom) irradiations are shown. Numbers of scans and irradiation condition (100% power) are indicated below.
(B) Schematic representation of Polη domains: UBZ, ubiquitin-binding motif-type zinc finger (residues 631–659); NLS, nuclear localization signal; PIP, domain of interaction with PCNA (residues 703–713) and Polη deletion (A, B, C, D, and F) or point (E and F) mutants. After irradiation, accumulation of Polη mutants at sites of DNA damage was analyzed as indicated: yes, accumulation in all scanned cells; no, no accumulation; +/−, accumulation in some but not all scanned cells. The X represents point mutation introduced in the UBZ of Polη sequence. Bottom panel: Representative images are shown for cells transfected with pGFP-Polη WT or UBZ-ΔPIP.
(C) Transformed XP30RO (XPV) cells stably expressing wild-type (WT) or mutated Polη (polDEAD, PIP∗, UBZ∗, UBZ∗/PIP∗) were treated with H2O2, then insoluble fractions or whole-cell extracts (WCE) were analyzed by western blot using indicated antibodies.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2011 Elsevier Inc. Terms and Conditions

9. Figure 7
Oxidative Stress Induces Persistent Monoubiquitination of PCNA in Nonreplicating XPV Fibroblasts
(A) Confluent primary fibroblasts AS405 (WT) or AS546 (XPV) were treated with 600 μM H2O2 and incubated for indicated times. Total extracts were then analyzed by immunoblotting using indicated antibodies.
(B) Transformed XPV cells (XP30RO) or XP30RO complemented with Polη WT (XP30ROPolη) were exposed to increasing doses of H2O2, and cell viability was counted 72 hr posttreatment. Percentage of control growth was plotted for each time point, representing the mean of three independent experiments (± SD).
(C) Primary fibroblasts AS405 (WT) or AS546 (XPV) were cultured at confluency as indicated in (A) and pretreated with aphidicolin 1 hr before H2O2 treatment. Cells were then incubated in fresh medium containing aphidicolin for indicated times. Total extracts were processed as in (A).
(D) MEFs preincubated with [3H]thymidine were exposed to increasing doses of H2O2, and DNA repair synthesis was measured 3 hr later. Cells were fixed and processed as described in the Supplemental Experimental Procedures.
(E) WT, POLH−/−, and MSH2−/− MEFs were pretreated with 10 μg/ml aphidicolin 2 hr before H2O2 exposure. They were then incubated for 3 hr in medium containing aphidicolin and [3H]thymidine before fixation. DNA repair synthesis was measured as described in the Supplemental Experimental Procedures. The graph represents the fold change in UDS measured by dividing the number of cells with ≥10 grains per nucleus after H2O2 treatment with the mock-treated ones.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2011 Elsevier Inc. Terms and Conditions