1. Interaction of Human DNA Polymerase η with Monoubiquitinated PCNA
Patricia L Kannouche, Jonathan Wing, Alan R Lehmann 
Molecular Cell 
Volume 14, Issue 4, Pages (May 2004)
DOI: /S (04)00259-X

2. Figure 1
PCNA Is Monoubiquitinated by DNA Damaging Agents that Block the Replication Forks
(A) SV40-transformed MRC5 fibroblasts were irradiated at 254 nm with 5 Jm−2 and incubated for 6 hr. (B) MRC5-Cl14 cells were transfected with pCDNA3.His-Ubiquitin. The following day, cells were either unirradiated (lane 3) or UV irradiated with 20 Jm−2 (lane 2) and incubated for 6 hr. Lane 1, untransfected control; lane 4, marker showing positions of PCNA species. (C) SV40-transformed fibroblasts were either unirradiated (−UV) or UV irradiated at 254 nm with 5 Jm−2 and incubated for the indicated times. (D) Cells were treated with the indicated dose of UV irradiation and incubated for 6 hr prior to harvesting. (E) MRC5 cells were treated with 1 mM hydroxyurea for 5 hr or 5 Gy γ-irradiation followed by incubation for the indicated times. SDS-PAGE was carried out on whole-cell extracts (A and C–E) or, after purification on nickel beads (B), followed by immunoblotting and probing with anti-PCNA antibody.
Molecular Cell  , DOI: ( /S (04)00259-X)

3. Figure 2
Ubi-PCNA and Polη Are Bound on the Chromatin after UV Irradiation
(A) MRC5-Cl14 cells either unirradiated or UV irradiated (20 Jm−2) were incubated for 6 hr, whole-cell extracts were extracted with Triton, and the Triton-insoluble fraction was solubilized. Equivalent amounts of whole-cell extract (WE), Triton-soluble fractions (SF), and insoluble fractions (IF) were loaded onto the gel. Immunoblot was performed with anti-polη or anti-PCNA antibodies.
(B) MRC5 cells transfected with a plasmid expressing eGFP-polη were fixed with paraformaldehyde (PFA) without (Ba and Bb) or following Triton extraction (Bc and Bd). Alternatively, cells expressing eGFP-polη were irradiated at 20 Jm−2, and 8 hr later, cells were treated with Triton, eGFP-polη was detected by autofluorescence (green staining, [Be]), and ubiquitinated proteins were revealed using anti-ubiquitin antibody (FK2) and TRITC-conjugated secondary antibody (red staining, [Bf]). Most of the polη foci colocalize with ubiquitinated proteins as shown in the right panel (Bg). MRC5-Cl14 cells were UV irradiated through a filter, and 8 hr later, cells were fixed and stained with anti-polη (Bh), anti-ubiquitin (FK2) (Bi), or DAPI (Bj). (Bk) shows the merged images.
(C) Unirradiated (lane 3) or UV-irradiated cells were incubated for 6 hr, followed by Triton extraction. The irradiated samples were incubated with (lane 1) or without (lane 2) DNase I. Immunoblots were carried out using anti-polη or anti-PCNA antibodies.
Molecular Cell  , DOI: ( /S (04)00259-X)

4. Figure 3 Ubi-PCNA Interacts Specifically with Polη
(A) UV-irradiated MRC5-Cl14 cells were incubated for 6 hr, Triton extracted, and crosslinked with formaldehyde. Triton-insoluble proteins were solubilized and immunoprecipitated with anti-PCNA and the immunoprecipitates analyzed by Western blotting with anti-polη (top) or anti-PCNA (bottom). Dashed arrow, nonspecific band.
(B) Cells transfected with pCDNA3.His-Ubiquitin were UV irradiated (lanes 1–4) or not (lane 5), incubated for 6 hr prior to Triton extraction, crosslinking (except lane 2), and harvesting. Resolubilized proteins were adsorbed on nickel-agarose, and the proteins remaining attached to the beads were analyzed by Western blotting using anti-PCNA or anti-polη antibodies. Lane 1 represents a sample of whole extracts from irradiated cells to indicate the positions of unmodified and ubiquitinated PCNA.
(C) Cells were either untransfected (lane 5) or transfected with pcDNA3 plasmid expressing His-tagged PCNA (lanes 2 and 3) or PCNA-K164R (lanes 1 and 4) and either unirradiated (lanes 1 and 2) or UV irradiated (lanes 3–5). After 6 hr, cells were harvested and then treated as in (B). (Ca) Immunoblots of nickel pull-downs probed with anti-PCNA or anti-polη antibodies. (Cb) Immunoblots of the total Triton-insoluble fractions probed with the same antibodies.
Molecular Cell  , DOI: ( /S (04)00259-X)

5. Figure 4 Ubiquitination of PCNA Is Dependent on hRad18
(A) Cells were transfected with a mixture of hRad18-specific siRNAs (lanes 1–4, 7, and 8) or cyclophilin siRNAs (lanes 5 and 6) at different concentrations and incubated for 72 hr. The cells were then either unirradiated (lanes 7 and 8) or UV irradiated and incubated for a further 6 hr. The cells were harvested and immunoblotted with anti-PCNA antibody.
(B) Cells were cotransfected with His-PCNA and hRad18, either wild-type (lane 1) or C28F mutant (lane 2), UV irradiated, and incubated for 6 hr. The cells were then harvested and extracts immunoblotted with anti-PCNA antibody.
Molecular Cell  , DOI: ( /S (04)00259-X)

6. Figure 5 Polη Interacts with Ubi-PCNA via Two Different Motifs
(A) MRC5 cells were transfected with peGFP-polη, peGFP-polηΔ705–713 (mutant A), peGFP-polηNFP-AAA (aa 324–326, mutant B), or with peGFP-polη containing both mutations (mutant AB), and 20 hr later they were UV irradiated (20 Jm−2). They were then incubated for a further 6 hr prior to Triton extraction, crosslinking, and harvesting. Triton-insoluble proteins probed with anti-polη (top) or anti-PCNA (bottom) are shown in (Ab). These were solubilized and immunoprecipitated with anti-PCNA antibody and the immunoprecipitates analyzed by Western blotting with anti-polη ([Aa], top) or anti-PCNA ([Aa], bottom). Dashed arrow, nonspecific band.
(B) Survival of XP30RO cells transfected with empty pCDNA3 vector or vector containing wild-type or AB mutant polη.
(C) Model. As discussed in our previous work, polη and polι are localized in replication factories and may or may not be associated with replication complexes. Monoubiquitination of PCNA (black ring) by the Rad6/Rad18 complex results in interaction between polη and PCNA, enabling TLS to take place. After bypassing the damage, polδ is switched in again, which may or may not require removal of the ubiquitin moiety from PCNA.
Molecular Cell  , DOI: ( /S (04)00259-X)