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, 7 (11), 1134-9

DNA Damage Induces Cdt1 Proteolysis in Fission Yeast Through a Pathway Dependent on Cdt2 and Ddb1

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DNA Damage Induces Cdt1 Proteolysis in Fission Yeast Through a Pathway Dependent on Cdt2 and Ddb1

Emma Ralph et al. EMBO Rep.

Erratum in

  • EMBO Rep. 2007 Feb;8(2):200

Abstract

Cdt1 is an essential protein required for licensing of replication origins. Here, we show that in Schizosaccharomyces pombe, Cdt1 is proteolysed in M and G1 phases in response to DNA damage and that this mechanism seems to be conserved from yeast to Metazoa. This degradation does not require Rad3 and Cds1, indicating that it is independent of classic DNA damage and replication checkpoint pathways. Damage-induced degradation of Cdt1 is dependent on Cdt2 and Ddb1, which are components of a Cul4 ubiquitin ligase. We also show that Cdt2 and Ddb1 are needed for cell-cycle changes in Cdt1 levels in the absence of DNA damage. Cdt2 and Ddb1 have been shown to be involved in the degradation of the Spd1 inhibitor of ribonucleotide reductase after DNA damage, and we speculate that Cdt1 downregulation might contribute to genome stability by reducing demand on dNTP pools during DNA repair.

Figures

Figure 1
Figure 1
DNA damage reduces Cdt1 levels in M- and G1-phase cells. (A) Western blot analysis of Cdt1–Myc levels after ultraviolet (UV) irradiation of nitrogen-starved G1-arrested cells (P1424). Cells used for lanes 2–6 were released from the block for 30 min by re-feeding at 34°C. (B) Western blot of Cdt1–Myc levels in the mitotic cell cycle after UV irradiation. nda3 cells (P1451) were arrested in mitosis, UV irradiated (100 J/m2) and then released from the block by shifting to 32°C. Log-phase extracts are shown for comparison. (C) Nuclear counts for cells in experiment shown in (B) showing the timing of anaphase. (D) Septation index of cells for experiment shown in (B). (E) Western blot analysis of Cdt1–Myc and Cdc18–TAP levels in mitotically arrested cells after UV irradiation (100 J/m2). Strains P1451 and P1452 were treated as in (B), except that cells were kept at 20°C after irradiation to maintain the mitotic arrest. (F) Nuclear counts of the experiment shown in (E), showing that UV irradiation does not lead to the release of cells from the nda3 mitotic block. (G) Cdt1–Myc levels after treatment with DNA damage and stress agents. Cells (P1451) were arrested at the nda3 block, treated with 10 μg/ml bleomycin (bleo), 0.2% MMS, 0.6 μg/ml 4-NQO, 0.5 mM hydrogen peroxide (H2O2), 32 μg/ml chloroquin (CQ), 0.5 mM CdSO4 or 1 M sorbitol, and extracts were prepared 60 min later. Cells were kept at 20°C to maintain the mitotic arrest. MMS, methyl methane sulphonate; 4-NQO, 4-nitroquinoline 1-oxide; TAP, tandem affinity purification tag.
Figure 2
Figure 2
Reduction in Cdt1 levels after ultraviolet irradiation is proteolysis dependent. Western blot of Cdt1–Myc in nda3 (P1451) cells that were arrested in mitosis at 20°C, UV irradiated (100 J/m2) and then incubated with or without cycloheximide (CHX; 100 μg/ml). Cells were kept at 20°C to maintain the mitotic arrest.
Figure 3
Figure 3
Ultraviolet-induced proteolysis of Cdt1 is not dependent on Rad3 and Cds1, but requires Ddb1 and Cdt2. (A) Cdt1–Myc levels after UV treatment in nda3 (wild type (wt), P1451) and nda3 rad3Δ cells (P1630) arrested at 20°C. (B) Cdt1–Myc levels after UV treatment in nda3 (wt, P1451) and nda3 cds1Δ (P1623) cells arrested at 20°C. (C) Cdt1–Myc levels after UV treatment in nda3 (wt, P1451) and nda3 ddb1Δ (P1615) cells arrested at 20°C. (D) Cdt1–Myc levels after UV treatment in nda3 (wt, P1451) and nda3 cdt2Δ (P1706) cells arrested at 20°C. In (AD), the mitotic arrest was maintained throughout the time course and all UV treatments were carried out 100 J/m2. (E) High-molecular-weight ubiquitylated Cdt1 levels are increased after UV irradiation in cdt2+ strains. Ubiquitylated proteins were purified under denaturing conditions from strains expressing His6–ubiquitin (His-Ub) by Ni2+-NTA agarose affinity chromatography and probed with peroxidase–anti-peroxidase soluble complex to detect Cdt1–TAP. Irradiated strains were exposed to UV (100 J/m2) and grown for 20 min before preparing extracts. Strains used were P137 (lane 1), P1517 (lane 2), P1783 (lanes 3,4) and P1785 (lanes 5,6). NTA, nitrilo-triacetic acid; TAP, tandem affinity purification tag.
Figure 4
Figure 4
Cdt2 and Ddb1 regulate Cdt1 levels during the mitotic cell cycle. (A) Cdt1-yellow fluorescent protein (YFP) imaged in asynchronous wild-type (wt; P1262), ddb1Δ (P1769) and cdt2Δ (P1674) cells. Scale bar, 10 μm. (B) Strains shown in (A) were analysed to determine the percentage of cells showing nuclear Cdt1–YFP (top) and the fluorescence levels of nuclear Cdt1–YFP (bottom). Cells were analysed in asynchronous culture (data are given for binucleate (late M/G1/S) and uninucleate (G2) cells) and after S-phase arrest (HU for 3 h). (C) Flow-cytometric analysis of cells analysed in (B). (D) Comparison of Cdt1 and Spd1 levels in wt (P1517, P1766), ddb1Δ(P1673, P1767), cdt2Δ (P1731, P1768) and mts3-1 (P1611) cell extracts made from asynchronous cultures. The mts3-1 strain was grown at a semi-permissive temperature. DAPI, 4,6-diamidino-2-phenylindole; HU, hydroxyurea; TAP, tandem affinity purification tag.

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