Regulation of proliferating cell nuclear antigen ubiquitination in mammalian cells - PubMed (original) (raw)

Regulation of proliferating cell nuclear antigen ubiquitination in mammalian cells

Atsuko Niimi et al. Proc Natl Acad Sci U S A. 2008.

Abstract

After exposure to DNA-damaging agents that block the progress of the replication fork, monoubiquitination of proliferating cell nuclear antigen (PCNA) mediates the switch from replicative to translesion synthesis DNA polymerases. We show that in human cells, PCNA is monoubiquitinated in response to methyl methanesulfonate and mitomycin C, as well as UV light, albeit with different kinetics, but not in response to bleomycin or camptothecin. Cyclobutane pyrimidine dimers are responsible for most of the PCNA ubiquitination events after UV-irradiation. Failure to ubiquitinate PCNA results in substantial sensitivity to UV and methyl methanesulfonate, but not to camptothecin or bleomycin. PCNA ubiquitination depends on Replication Protein A (RPA), but is independent of ATR-mediated checkpoint activation. After UV-irradiation, there is a temporal correlation between the disappearance of the deubiquitinating enzyme USP1 and the presence of PCNA ubiquitination, but this correlation was not found after chemical mutagen treatment. By using cells expressing photolyases, we are able to remove the UV lesions, and we show that PCNA ubiquitination persists for many hours after the damage has been removed. We present a model of translesion synthesis behind the replication fork to explain the persistence of ubiquitinated PCNA.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

PCNA ubiquitination after DNA-damaging agents. (A and B) MRC5V1 cells were UV-irradiated with 10 (A) or 20 (B) J m−2, incubated for the indicated times, and analyzed by immunoblotting with anti-PCNA (Upper) or anti-USP1 antibody (Lower). −UV, mock-treated cells incubated for 6 h. (C) XP-A cells expressing the indicated photolyase were UV-irradiated (10 J m−2), exposed or not to photoreactivating light for 2 h (PR), incubated for a further 6 h, and analyzed as in A. (D and E) Cells were treated with 1 mM MMS for 1h (D) or 8 μg/ml mitomycin C for 30 min (E), followed by incubation for the indicated times before harvesting and analysis by immunoblotting.

Fig. 2.

Fig. 2.

UV sensitivity of cells expressing PCNA-K164R. (A) MRC5V1 cell clones expressing His-tagged wild-type PCNA or PCNA-K164R were either mock-transfected or treated with PCNA-specific siRNA and PCNA levels measured after 72 h. (B) After transfection with PCNA siRNA, the cells were UV-irradiated (20 J m−2), incubated for 5 h, and analyzed for PCNA ubiquitination. (C–E) UV, MMS, and camptothecin survival curves of cells depleted for endogenous PCNA and expressing wild-type (WT) or mutant (KR) His-PCNA. Where indicated (-h), cells were also depleted for polη. Error bars, ±SEM of three or four experiments.

Fig. 3.

Fig. 3.

PCNA ubiquitination in RPA or ATR knockdown cells. (A) MRC5V1 cells were transfected with RPA70 siRNA. After a 72-h incubation, cells were either irradiated or not with 10 J m−2 UV and incubated for 0.5 or 6 h. Cell extracts were analyzed by immunoblotting with (Top to Bottom) anti-RPA70, anti-Chk1-P-Ser-317, PC10, and anti-vimentin (loading control) antibody. Lanes 1, 3, and 5, nontargeting siRNA control. Lanes 2, 4, and 6, RPA siRNA-transfected samples. (B) MRC5V1 cells were transfected with nontargeting or ATR siRNA, UV-irradiated (20 J m−2) 72 h later, incubated for 6 h, and analyzed as in A. (C) Normal or Seckel syndrome lymphoblastoid cells were UV-irradiated with the indicated doses and incubated for 6 h before lysis and analysis.

Fig. 4.

Fig. 4.

Persistence of PCNA ubiquitination. PH-XPA cells were irradiated with 20 J m−2 UV, incubated for 6 h, and then photoreactivated for 2 h. After further incubation for the indicated times, PCNA in cell lysates was detected by immunoblotting. In A, duplicate samples were analyzed either with or without prior extraction with Triton X-100, as indicated. (B) Lysates were analyzed for both USP1 and PCNA ubiquitination.

Fig. 5.

Fig. 5.

Model for persistence of Ub-PCNA. (A) On blocking of the replication fork at a lesion (X), PCNA becomes ubiquitinated (U). (Note that only one ubiquitin molecule is shown for simplicity, but it is likely that all three monomers of the homotrimeric ring become ubiquitinated.) (B) A new replication apparatus is assembled beyond the lesion, leaving a gap. (C) The process is repeated at the next lesion. (D) Some time later, the gap opposite the first lesion is filled, as indicated by the thick line.

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