Postreplication repair and PCNA modification in Schizosaccharomyces pombe - PubMed (original) (raw)

Postreplication repair and PCNA modification in Schizosaccharomyces pombe

Jonathan Frampton et al. Mol Biol Cell. 2006 Jul.

Abstract

Ubiquitination of proliferating cell nuclear antigen (PCNA) plays a crucial role in regulating replication past DNA damage in eukaryotes, but the detailed mechanisms appear to vary in different organisms. We have examined the modification of PCNA in Schizosaccharomyces pombe. We find that, in response to UV irradiation, PCNA is mono- and poly-ubiquitinated in a manner similar to that in Saccharomyces cerevisiae. However in undamaged Schizosaccharomyces pombe cells, PCNA is ubiquitinated in S phase, whereas in S. cerevisiae it is sumoylated. Furthermore we find that, unlike in S. cerevisiae, mutants defective in ubiquitination of PCNA are also sensitive to ionizing radiation, and PCNA is ubiquitinated after exposure of cells to ionizing radiation, in a manner similar to the response to UV-irradiation. We show that PCNA modification and cell cycle checkpoints represent two independent signals in response to DNA damage. Finally, we unexpectedly find that PCNA is ubiquitinated in response to DNA damage when cells are arrested in G2.

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Figures

Figure 1.

Figure 1.

In vitro ubiquitination reactions. (A) Ubiquitination reactions were carried out with E1, recombinant S. pombe Ubc13 and Mms2 and either wild-type or lysine mutants of ubiquitin, and reactions were incubated for 0, 2, or 4 h, as indicated. Reaction products were analyzed by immunoblotting with anti-ubiquitin antibody (note that the apparently anomalous migration in lanes 4–6 resulted from a bubble during transfer and was not reproduced in other experiments). (B) Cross-species interactions of Ubc13 and Mms2 from S. pombe (S.p.) and S. cerevisiae (S.c.). Reactions carried out as in A. Ub, Ub2, Ub3, Ub4: mono-, di-, tri-, and tetra-ubiquitin.

Figure 2.

Figure 2.

Ubiquitination of PCNA in S. pombe in response to DNA damage. (A) Exponentially growing cultures of S. pombe were exposed to 50 Jm−2 UV and incubated for 30 min or to 30 μM camptothecin (CPT), 0.9 mM MMS, or 50 mM HU for 3 h. The cells were then lysed and analyzed for PCNA modification using anti-PCNA antibody. Ub, Ub2, Ub3: mono-, di-, and triubiquitinated PCNA. (B) Cells were transfected with pMHRep41 either as empty vector or expressing Myc2His6-tagged ubiquitin and then incubated with or without HU for 3 h. SDS lysates were incubated with Ni-agarose beads, and the bound proteins were analyzed with anti-PCNA antibody. MHUb1, MHUb2: PCNA modified with one or two myc-his–tagged ubiquitins. The asterisk represents a nonspecific cross-reacting band. Note that a small amount of unmodified PCNA (<1% of the total PCNA) bound nonspecifically to the beads in all lanes. (C) The indicated mutants were either untreated (−) or exposed to 50 Jm−2 UV-irradiation (+) and incubated for 30 min before harvesting and analysis as in A. Mono-, di-, and triubiquitinated PCNA bands are indicated with arrowheads. The fainter bands are nonspecific cross-reacting species.

Figure 3.

Figure 3.

Epistasis analysis. (A–C) UV survival curves of single and double mutants of _mms2_Δ with (A) ubc13 Δ, (B) rhp18 Δ, and (C) rad8 Δ. (D–F) UV survival curves of single and double mutants of pcn1-K164R with (D) _rhp18_Δ, (E) rad8Δ, and (F) _mms2_Δ. Means ± SEM of three experiments are shown. Double mutants are indicated with a bold line.

Figure 4.

Figure 4.

PCNA ubiquitination and cell cycle checkpoints are independent responses. (A) Checkpoint mutants were UV-irradiated (50 Jm−2) and analyzed for PCNA ubiquitination. Arrowheads indicate ubiquitinated species. (B) Chk1 is activated in pcn1-K164R after exposure to UV. Cultures of exponentially growing wild-type, pcn1-K164R and rad9-T412A cells, both containing an integrated single copy of HA-tagged chk1, were exposed to 100 Jm−2 UV radiation and then incubated for 30 min. Total cell extracts were analyzed by Western blotting with anti-HA antibodies. Arrowheads indicate activated Chk1. (C) UV survival curves of single and double mutants of pcn1-K164R with _rad3_Δ.

Figure 5.

Figure 5.

PCNA ubiquitination in response to ionizing radiation. (A) The indicated mutants were exposed to 500 Gy ionizing radiation and incubated for 1 h. PCNA modification was analyzed in whole cell extracts by immunoblotting. Arrowheads indicate ubiquitinated PCNA bands; note that in this experiment there was a strongly cross-reacting nonspecific band in all samples, indicated with an asterisk (*). (B–D) IR survival curves of single and double mutants of _mms2_Δ with (B) ubc13 Δ, (C) rhp18 Δ, and (D) rad8Δ. (E and F) UV and IR survival curves, respectively, of single and double mutants of pcn1-K164R with _rhp51_Δ. Double mutants indicated with bold lines.

Figure 6.

Figure 6.

PCNA ubiquitination through the cell cycle. Cells synchronized by elutriation were either mock-irradiated (A) or UV-irradiated (50 Jm−2; C) at different times after reinoculation and then incubated for 30 min. PCNA modification was analyzed by immunoblotting of whole cell extracts. (B) Cell cycle position was analyzed by microscopic measurement of mitotic index and septation index 30 min after UV-irradiation (UV-treated) or mock treatment (untreated). Note that septation occurs in early S phase in S. pombe.

Figure 7.

Figure 7.

PCNA ubiquitination in G2. (A) cdc25.22 cells were maintained either at 25°C (asynchronous) or 36°C (G2) for 3 h. Cells were then treated with either 50 mM HU or 50 μM 4NQO for 1 h or UV-irradiated (100 Jm−2) and incubated for 60 min. (B) _cdc25.22 rad13Δ uve1_Δ or _cdc25.22 rad3Δ tel1_Δ triple mutants were treated at as in A. In all cases PCNA modification was analyzed by immunoblotting of whole cell extracts.

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