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. 2006 Dec 4;175(5):703-8.
doi: 10.1083/jcb.200606145. Epub 2006 Nov 27.

Human SHPRH suppresses genomic instability through proliferating cell nuclear antigen polyubiquitination

Akira Motegi  1 , Raman Sood, Helen Moinova, Sanford D Markowitz, Pu Paul Liu, Kyungjae Myung
Affiliations

Human SHPRH suppresses genomic instability through proliferating cell nuclear antigen polyubiquitination

Akira Motegi et al. J Cell Biol. .

Abstract

Differential modifications of proliferating cell nuclear antigen (PCNA) determine DNA repair pathways at stalled replication forks. In yeast, PCNA monoubiquitination by the ubiquitin ligase (E3) yRad18 promotes translesion synthesis (TLS), whereas the lysine-63-linked polyubiquitination of PCNA by yRad5 (E3) promotes the error-free mode of bypass. The yRad5-dependent pathway is important to prevent genomic instability during replication, although its exact molecular mechanism is poorly understood. This mechanism has remained totally elusive in mammals because of the lack of apparent RAD5 homologues. We report that a putative tumor suppressor gene, SHPRH, is a human orthologue of yeast RAD5. SHPRH associates with PCNA, RAD18, and the ubiquitin-conjugating enzyme UBC13 (E2) and promotes methyl methanesulfonate (MMS)-induced PCNA polyubiquitination. The reduction of SHPRH by stable short hairpin RNA increases sensitivity to MMS and enhances genomic instability. Therefore, the yRad5/SHPRH-dependent pathway is a conserved and fundamental DNA repair mechanism that protects the genome from genotoxic stress.

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Figures

Figure 1.
Figure 1.
Human SHPRH and yeast Rad5 share the domain structure. (A) Differential modifications of PCNA determine DNA repair pathways. R18, Rad18; R5, Rad5; R6, Rad6; 13, Ubc13; 2, Mms2. (B) Schematic representation of human (Hs) SHPRH and yeast (Sc) Rad5. SWI2/SNF2 (subdomains I, Ia, II, III, IV, V, and VI) and RING finger domains are indicated. Multiple alignments of RING finger domains of human (Hs), rat (Rn), mouse (Mm), zebrafish (Dr) SHPRH homologues, fission yeast (Sp) rad8 (a Rad5 homologue), and budding yeast (Sc) Rad5. Dots indicate the conserved cysteines and histidine. The cysteine mutated in this study (C1432) is indicated with an asterisk.
Figure 2.
Figure 2.
SHPRH is a functional orthologue of yeast Rad5. (A) SHPRH promotes PCNA polyubiquitination. HEK 293T cells were transfected with plasmids expressing 0.5 μg HA-ubiquitin (HA-Ub), 0.5 μg FLAG-PCNA, 2.0 μg SHPRH-myc-His, 100 ng RAD6-HA, 50 ng UBC13-HA, 50 ng UBC13(C87A)-HA, and 50 ng MMS2-HA in the combinations indicated. Lysates were immunoprecipitated with an anti-FLAG antibody, and ubiquitinated PCNAs were detected by an anti-HA antibody. Total PCNAs were probed with an anti-FLAG antibody. PCNA-Ub1 and PCNA-UbN indicate mono- and polyubiquitinated PCNAs, respectively. Mono- and diubiquitinated PCNAs are marked with single and double asterisks, respectively. Expression of SHPRH and E2s was analyzed by blotting lysates with specific anti-epitope tag antibodies. (B) E3 ligase activity of SHPRH for PCNA polyubiquitination is perturbed by a mutation in the conserved cysteine of the RING domain (C1432A). (C) HEK 293T cells were transfected as in A, except using ubiquitin mutants (left), PCNA-K164R mutant (middle), or lentivirus-infected 293T cells (right). The dot represents the IgG heavy chain. Lanes 4 and 5 of the top panel were exposed longer than lanes 6 and 7. (D) RAD18 and SHPRH cooperate to promote PCNA polyubiquitination. Cells were transfected as indicated with the same amounts of plasmids as in A, and endogenous PCNA was immunoprecipitated with an anti-PCNA antibody.
Figure 3.
Figure 3.
SHPRH physically interacts with PCNA, UBC13, and RAD18 and multimerizes. (A and B) SHPRH associates with PCNA and UBC13 in vitro. SHPRH-FLAG was pulled down with GST-fused PCNA (wild type or K164R mutant; A) or with GST-fused E2s (B) and analyzed by blotting with an anti-FLAG antibody. RAD18-FLAG was also pulled down with GST-PCNA (A). (C and D) SHPRH associates with UBC13 in vivo. SHPRH-myc-His (C) or SHPRH-FLAG (D) was coexpressed with wild-type UBC13-HA, UBC13(CA)-HA, or MMS2-HA in the combinations indicated. Anti-myc (C) or anti-HA (D) immunoprecipitates were blotted with an anti-HA (C) or anti-FLAG (D) antibody. Note that the level of SHPRH is enhanced in the presence of wild-type UBC13 and, to a lesser extent, UBC13(CA). SHPRH showed a weak but reproducible interaction with UBC13(CA). Blots with anti–α-tubulin or anti-GFP serve as loading or transfection control, respectively. (E and F) SHPRH interacts with RAD18 and self-multimerizes in vivo. In F, cell lysates expressing SHPRH-FLAG and SHPRH-myc-His were immunoprecipitated with anti-myc antibody and blotted with an anti-FLAG antibody. Normal IgG antibody was used for control.
Figure 4.
Figure 4.
PCNA polyubiquitination is induced by MMS. (A) HEK 293T cells transfected with plasmids expressing 0.2 μg HA-ubiquitin (Ub) and 0.2 μg FLAG-PCNA with or without 0.6 μg SHPRH-myc-His were treated with 30 J/m2 UV, 0.3 mM MMC, or 0.01% MMS for 2 h. Anti-FLAG immunoprecipitated PCNA was analyzed with an anti-HA antibody. (B) MMS treatment induces polyubiquitination of endogenous PCNA. HCT116 cells were treated with the indicated doses of MMS for 2 h. Immunoprecipitated PCNA was analyzed with an anti-ubiquitin (P4D1) antibody. (C) HCT116 cells in G1 (lanes 1 and 2) or S phase (lanes 3 and 4) were treated with 0.01% MMS for 2 h. A polyubiquitinated species of PCNA was detected with anti-polyubiquitin (FK2) antibody.
Figure 5.
Figure 5.
SHPRH suppresses MMS-induced genomic instability. (A) Expression level of SHPRH in individual clones carrying an integrated SHPRH-shRNA vector was analyzed by RT-PCR (top) and Western blot with anti-SHPRH antibody (bottom). (B) SHPRH-silenced cells show a reduced level of MMS-induced PCNA polyubiquitination. HCT116 cells with control (vector) or SHPRH-silencing (shRNA) lentivirus were treated with 0.01% MMS for 2 h. (C) The reduced expression of SHPRH sensitizes cells to MMS. Three independent shRNA clones were treated with increasing concentrations of MMS, and serially diluted cells were plated. Surviving colonies were counted and quantified. Doses of MMS for 10% survival are 0.0145% (wild type [wt]), 0.0110% (B2), 0.0090% (C4), and 0.0085% (B11). (D and E) Reduction of SHPRH enhances chromosome breaks by MMS. Metaphase chromosomes were analyzed 24 h after a 1-h treatment of different clones with 0.01% MMS. (D) One example from each clone is shown. (E) Noticeable chromosome breakages per cell were quantified from 100 metaphases per each clone.
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