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Ddb1 Is Required for the Proteolysis of the Schizosaccharomyces pombe Replication Inhibitor Spd1 during S Phase and after DNA Damage

2004; Elsevier BV; Volume: 279; Issue: 11 Linguagem: Inglês

10.1074/jbc.m312570200

1083-351X

Tanya Bondar, Aleksandr Ponomarev, Pradip Raychaudhuri,

Microtubule and mitosis dynamics

Recently we showed that the Schizosaccharomyces pombe ddb1 gene plays a role in S phase progression. A mutant S. pombe strain lacking expression of the ddb1 gene exhibited slow replication through both early and late regions causing a slow S phase phenotype. We attributed the phenotypes in the ddb1 strain to an increased activity of the replication checkpoint kinase Cds1. However, the basis for a high basal Cds1 activity in the ddb1 strain was not clear. It was shown that Ddb1 associates with the Cop9/signalosome. Moreover, the phenotypes of the Δddb1 strain are remarkably similar to the Δcsn1 (or Δcsn2) strain that lacks expression of the Csn1 (or Csn2) subunit of the Cop9/signalosome. Cop9/signalosome cooperates with Pcu4 to induce proteolysis of Spd1, which inhibits DNA replication by inhibiting ribonucleotide reductase. Therefore, we investigated whether Ddb1 is required for the proteolysis of Spd1. Here we show that a S. pombe strain lacking expression of Ddb1 fails to induce proteolysis of Spd1 in S phase and after DNA damage. Moreover, deletion of the spd1 gene attenuates the Cds1 kinase activity in cells lacking the expression of ddb1, suggesting that an accumulation of Spd1 results in the increase of Cds1 activity in the Δddb1 strain. In addition, the double mutant lacking spd1 and ddb1 no longer exhibits the growth defects and DNA damage sensitivity observed in the Δddb1 strain. Our results establish an essential role of Ddb1 in the proteolysis of Spd1. In addition, the observation provides evidence for a functional link between Ddb1 and the Cop9/signalosome. Recently we showed that the Schizosaccharomyces pombe ddb1 gene plays a role in S phase progression. A mutant S. pombe strain lacking expression of the ddb1 gene exhibited slow replication through both early and late regions causing a slow S phase phenotype. We attributed the phenotypes in the ddb1 strain to an increased activity of the replication checkpoint kinase Cds1. However, the basis for a high basal Cds1 activity in the ddb1 strain was not clear. It was shown that Ddb1 associates with the Cop9/signalosome. Moreover, the phenotypes of the Δddb1 strain are remarkably similar to the Δcsn1 (or Δcsn2) strain that lacks expression of the Csn1 (or Csn2) subunit of the Cop9/signalosome. Cop9/signalosome cooperates with Pcu4 to induce proteolysis of Spd1, which inhibits DNA replication by inhibiting ribonucleotide reductase. Therefore, we investigated whether Ddb1 is required for the proteolysis of Spd1. Here we show that a S. pombe strain lacking expression of Ddb1 fails to induce proteolysis of Spd1 in S phase and after DNA damage. Moreover, deletion of the spd1 gene attenuates the Cds1 kinase activity in cells lacking the expression of ddb1, suggesting that an accumulation of Spd1 results in the increase of Cds1 activity in the Δddb1 strain. In addition, the double mutant lacking spd1 and ddb1 no longer exhibits the growth defects and DNA damage sensitivity observed in the Δddb1 strain. Our results establish an essential role of Ddb1 in the proteolysis of Spd1. In addition, the observation provides evidence for a functional link between Ddb1 and the Cop9/signalosome. The Schizosaccharomyces pombe Ddb1 protein is homologous to the DDB1 subunit of the mammalian damaged DNA-binding protein DDB, 1The abbreviations used are: DDB, damaged DNA-binding protein; FACS, fluorescence-activated cell sorter; GST, glutathione S-transferase; HU, hydroxyurea; MOPS, 4-morpholinepropanesulfonic acid; RNR, ribonucleotide reductase; MBP, myelin basic protein.1The abbreviations used are: DDB, damaged DNA-binding protein; FACS, fluorescence-activated cell sorter; GST, glutathione S-transferase; HU, hydroxyurea; MOPS, 4-morpholinepropanesulfonic acid; RNR, ribonucleotide reductase; MBP, myelin basic protein. which contains an additional subunit DDB2 (1Hwang B.J. Chu G. Biochemistry. 1993; 32: 1657-1666Crossref PubMed Scopus (94) Google Scholar). The mammalian DDB has been implicated in global genomic repair (1Hwang B.J. Chu G. Biochemistry. 1993; 32: 1657-1666Crossref PubMed Scopus (94) Google Scholar, 2Abramic M. Levine A.S. Protic M. J. Biol. Chem. 1991; 266: 22439-22500Abstract Full Text PDF Google Scholar, 3Chu G. Chang E. Science. 1988; 242: 564-567Crossref PubMed Scopus (350) Google Scholar). The DDB2 subunit, which is not found in S. pombe or other lower organisms, is critical for recognition of damaged DNA and the global genomic repair functions of DDB (4Otrin V.R. Kuraoka I. Nardo T. McLenigan M. Eker A.P.M. Stefanini M. Levine A.S. Wood R.D. Mol. Cell. Biol. 1998; 18: 3182-3190Crossref PubMed Google Scholar). The S. pombe Ddb1 protein has been shown to be required for normal cell growth, progression through the S phase, and proper chromosome segregation (5Zolezzi F. Fuss J. Uzawa S. Linn S. J. Biol. Chem. 2002; 277: 41183-41191Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 6Bondar T. Mirkin E.V. Ucker D.S. Walden W.E. Mirkin S.M. Raychaudhuri P. J. Biol. Chem. 2003; 278: 37006-37014Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). Although it is not clear whether those functions are conserved in the mammalian DDB1 protein, both mammalian and yeast Ddb1 have been shown to associate with Cop9/signalosome, a large complex with homology to the 19 S lid complex of the proteasome (7Groisman R. Polanowska J. Kuraoka I. Sawada J. Saijo M. Drapkin R. Kisselev A.F. Tanaka K. Nakatani Y. Cell. 2003; 113: 357-367Abstract Full Text Full Text PDF PubMed Scopus (589) Google Scholar, 8Liu C. Powell K.A. Mundt K. Wu L. Carr A.M. Caspari T. Genes Dev. 2003; 9: 1130-1140Crossref Scopus (158) Google Scholar). The interaction of the mammalian DDB with the signalosome has been linked to repair, whereas the interaction of the yeast Ddb1 with signalosome has not been characterized. Cop9/signalosome was identified as a regulator of photomorphogenesis in plants (for review, see Ref. 9Cope G.A. Deshaies R.J. Cell. 2003; 114: 663-671Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar). It was shown to control the levels of the plant transcription factors Hy5 and HyH through subcellular localization and proteolysis (10Osterlund M.T. Ang L.H. Deng X.W. Trends Cell Biol. 1999; 9: 113-118Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). The plant signalosome complex associates with the E2 conjugating enzyme Cop1 and E3 ubiquitin ligase that are involved in the proteolysis of the Hy5 and HyH transcription factors depending upon the availability of light. Signalosome has been characterized also from mammals and fission yeast (11Mundt K.E. Porte J. Murray J.M. Brikos C. Christensen P.U. Caspari T. Hagan I.M. Millar J.B. Simanis V. Hofmann K. Carr A.M. Curr. Biol. 1999; 9: 1427-1430Abstract Full Text Full Text PDF PubMed Google Scholar, 12Seeger M. Kraft R. Ferrell K. Bech-Otschir D. Dumdey R. Schade R. Gordon C. Naumann M. Dubiel W. FASEB J. 1998; 12: 469-478Crossref PubMed Scopus (309) Google Scholar). Like the plant signalosome, the mammalian and the yeast signalosomes are involved in controlling proteolysis mediated by the cullin family of the E3 ubiquitin ligases. All cullins are modified by neddylation (NEDD8 conjugation), which is believed to be required for their ubiquitin ligase activity (13Lammer D. Mathias N. Laplaza J.M. Jiang W. Liu Y. Callis J. Goebl M. Estelle M. Genes Dev. 1998; 12: 914-926Crossref PubMed Scopus (278) Google Scholar, 14Liakopoulos D. Doenges G. Matuschewski K. Jentsch S. EMBO J. 1998; 17: 2208-2214Crossref PubMed Scopus (306) Google Scholar, 15Kawakami T. Chiba T. Suzuki T. Iwai K. Yamanaka K. Minato N. Suzuki H. Shimbara N. Hidaka Y. Osaka F. EMBO J. 2001; 20: 4003-4012Crossref PubMed Scopus (271) Google Scholar). Signalosome possesses a deneddylating activity that is able to remove the NEDD8 conjugation from the cullins (16Lyapina S. Cope G. Shevchenko A. Serino G. Tsuge T. Zhou C. Wolf D.A. Wei N. Deshaies R.J. Science. 2001; 292: 1382-1385Crossref PubMed Scopus (555) Google Scholar). The cullin deneddylation function of the signalosome was confirmed using both biochemical and genetic approaches (8Liu C. Powell K.A. Mundt K. Wu L. Carr A.M. Caspari T. Genes Dev. 2003; 9: 1130-1140Crossref Scopus (158) Google Scholar, 16Lyapina S. Cope G. Shevchenko A. Serino G. Tsuge T. Zhou C. Wolf D.A. Wei N. Deshaies R.J. Science. 2001; 292: 1382-1385Crossref PubMed Scopus (555) Google Scholar, 17Zhou C. Seibert V. Geyer R. Rhee E. Lyapina S. Cope G. Deshaies R.J. Wolf D.A. BMC Biochemistry.http://www.biomedcentral.com/2001/2/7-12Date: 2001Google Scholar, 18Yang X. Menon S. Lykke-Andersen K. Tsuge T. Di X. Wang X. Rodriguez-Suarez R.J. Zhang H. Wei N. Curr. Biol. 2002; 12: 667-672Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). For example, it was shown that the fission yeast mutant lacking expression of any of the signalosome subunits Csn1, Csn2, Csn3, Csn4, and Csn5 exhibited accumulation of the neddylated form of Pcu1 and Pcu3 (16Lyapina S. Cope G. Shevchenko A. Serino G. Tsuge T. Zhou C. Wolf D.A. Wei N. Deshaies R.J. Science. 2001; 292: 1382-1385Crossref PubMed Scopus (555) Google Scholar, 17Zhou C. Seibert V. Geyer R. Rhee E. Lyapina S. Cope G. Deshaies R.J. Wolf D.A. BMC Biochemistry.http://www.biomedcentral.com/2001/2/7-12Date: 2001Google Scholar). Surprisingly, however, only the Δcsn1 and the Δcsn2 mutants exhibited a phenotype, suggesting the existence of additional functions of those two subunits of the S. pombe signalosome (11Mundt K.E. Porte J. Murray J.M. Brikos C. Christensen P.U. Caspari T. Hagan I.M. Millar J.B. Simanis V. Hofmann K. Carr A.M. Curr. Biol. 1999; 9: 1427-1430Abstract Full Text Full Text PDF PubMed Google Scholar). Recent studies on the function of the S. pombe Csn1 and Csn2 subunits revealed their role in positively regulating the activity of the ribonucleotide reductase (RNR) through a proteolysis of the inhibitor Spd1 (8Liu C. Powell K.A. Mundt K. Wu L. Carr A.M. Caspari T. Genes Dev. 2003; 9: 1130-1140Crossref Scopus (158) Google Scholar). Spd1 retains the Suc22 subunit of RNR in the nucleus. The periodic proteolysis of Spd1 in early S phase releases Suc22, which associates with Cdc22 in the cytoplasm to reconstitute the active RNR that is required for the biosynthesis of dNTPs (8Liu C. Powell K.A. Mundt K. Wu L. Carr A.M. Caspari T. Genes Dev. 2003; 9: 1130-1140Crossref Scopus (158) Google Scholar). It was shown that the Csn1 subunit of signalosome cooperated with Pcu4 to enhance the proteolysis of Spd1 (8Liu C. Powell K.A. Mundt K. Wu L. Carr A.M. Caspari T. Genes Dev. 2003; 9: 1130-1140Crossref Scopus (158) Google Scholar). The proteolysis of Spd1 occurs also during DNA damage requiring the activation of the checkpoint pathway (8Liu C. Powell K.A. Mundt K. Wu L. Carr A.M. Caspari T. Genes Dev. 2003; 9: 1130-1140Crossref Scopus (158) Google Scholar). It was shown that the checkpoint-dependent proteolysis of Spd1 involved Csn1 because the S. pombe strains lacking this gene failed to induce proteolysis of Spd1 after ionizing radiation (8Liu C. Powell K.A. Mundt K. Wu L. Carr A.M. Caspari T. Genes Dev. 2003; 9: 1130-1140Crossref Scopus (158) Google Scholar). These observations established a positive role of the S. pombe signalosome in the ubiquitin-proteasome-mediated proteolysis of Spd1. Moreover, it was shown that mutation of Spd1 caused a reversal of many of the growth-related phenotypes of the S. pombe strains lacking expression of csn1 (8Liu C. Powell K.A. Mundt K. Wu L. Carr A.M. Caspari T. Genes Dev. 2003; 9: 1130-1140Crossref Scopus (158) Google Scholar, 11Mundt K.E. Porte J. Murray J.M. Brikos C. Christensen P.U. Caspari T. Hagan I.M. Millar J.B. Simanis V. Hofmann K. Carr A.M. Curr. Biol. 1999; 9: 1427-1430Abstract Full Text Full Text PDF PubMed Google Scholar). Our recent studies on a S. pombe strain lacking expression of Ddb1 exhibited a remarkable overlap of the phenotypes that were described for the strains lacking expression of the Csn1 or Csn2 subunits of the signalosome. As in the case of the Δcsn1 or Δcsn2 strain, the Δddb1 strain exhibited slow growth and extended S phase. We showed that there was a delay in the progression of DNA synthesis in the Δddb1 strain (6Bondar T. Mirkin E.V. Ucker D.S. Walden W.E. Mirkin S.M. Raychaudhuri P. J. Biol. Chem. 2003; 278: 37006-37014Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). Moreover, the Δddb1 strain was hypersensitive to DNA damage in S phase and failed to recover from hydroxyurea (HU) block (6Bondar T. Mirkin E.V. Ucker D.S. Walden W.E. Mirkin S.M. Raychaudhuri P. J. Biol. Chem. 2003; 278: 37006-37014Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). Further, as in the case of the Δcsn1 strain, the Δddb1 strain exhibited a high level of the active Cds1 kinase. Because of these overlaps in the phenotypes, we considered the possibility that Ddb1 might be involved in the same pathway with signalosome to regulate Spd1 during S phase and after DNA damage. Here we show that Ddb1 is an essential component in the proteolysis of Spd1. The S. pombe strain lacking expression of Ddb1 fails to induce proteolysis of Spd1 during the cell cycle and after DNA damage or replicative stress. Moreover, mutation in the Spd1 gene reverses many of the defects that were observed in the Δddb1 strain. The observations provide evidence for a functional link between Ddb1 and signalosome in the cell cycle- and checkpoint-regulated proteolysis of the replication inhibitor Spd1. Yeast Cultures—Cells were cultured in yeast extract plus supplements (YE5S) medium at 32 °C. Temperature-sensitive strains were cultured at room temperature (22 °C) and synchronized by a shift to 35 °C for 3 h. Genetic crosses were performed on EMM-glutamate plates. The double mutant strains were constructed by random spore analysis followed by selection on appropriate media. Genotypes were then confirmed by genomic PCR (Table I).Table IStrain listStrainGenotypeSourceARC 556h + ade6-M216 ura4-D18leu1-32Paul NurseFY 319h-ade6-M216 ura4-D18 leu1-32 can1-1 cdc25-22Susan ForsburgTB 13-1h+ ade6-M216 ura4-D18 leu1-32 ddb1::kanrOur stockTB 16h+ ade6-M216 ura4-D18 leu1-32 cdc25-22 ddb1::kanrOur stockKP 38h- ade6-704 ura4-D18 leu1-32 spd1::ura4Antony CarrTB 31-1h90 ade6-704 ura4-D18 leu1-32 ddb1::kanr spd1::ura4This studyh- spd1-GSTPaul NurseTB 36-21h+ ddb1::kanr spd1:GSTThis study Open table in a new tab UV Irradiation—For the UV sensitivity experiments, equal aliquots of cells were plated on YE5S agar in triplicate and irradiated with 0–200 J/m2 UV-C at a rate of 30 J/m2/s. Colonies were counted after incubation at 32 °C for 3 days. Survival is expressed as a percentage of colonies observed on the mock-treated plates. For the Spd1 degradation assay, cells grown in YE5S were collected on a Millipore filter, irradiated with 450 J/m2 UV-C, and resuspended in the original media. Aliquots were removed for Western blotting analysis immediately before or 20 and 40 min after irradiation. Protein Extracts and Western Blotting—For protein extracts, cells were washed with STOP buffer (150 mm NaCl, 50 mm NaF, 10 mm EDTA, 1 mm NaN3, pH 8.0), resuspended in HB buffer (25 mm MOPS, pH 7.2, 15 mm MgCl2, 15 mm EGTA, 1% Triton X-100, 80 mm β-glycerophosphate, 15 mm p-nitrophenyl phosphate, 0.1% Nonidet P-40, 5 mm EDTA, 1 mm dithiothreitol, 0.1 mm sodium vanadate, 1 mm phenylmethylsulfonyl fluoride, 20 μg/ml leupeptin, 40 μg/ml aprotinin), and broken open by vortexing with glass beads at 4 °C. The extract was cleared by centrifugation. The protein concentration was estimated by the Bradford method. Extracts were boiled in SDS loading buffer and separated on SDS-PAGE. Proteins were transferred to nitrocellulose membrane. After blocking with 5% dry milk in PBS containing 0.1% Nonidet P-40, the membrane was probed with anti-Cds1 antibody (a kind gift from Teresa Wang, Stanford University), anti-GST, anti-ubiquitin (Santa Cruz), or anti-Spd1 antibody (kindly provided by Paul Nurse, Cancer Research UK) followed by an appropriate secondary horseradish peroxidase-conjugated antibody. For anti-Spd1 and anti-ubiquitin Western blotting, the membrane was autoclaved prior to blocking; the anti-Spd1 antibody was precleared by incubation with nitrocellulose to adsorb background signal. The signal was visualized by ECL (Amersham Biosciences). Pulse-Chase—Cells were growth into log phase in YE5S, span down, and resuspended in YE5S without methionine at 2 × 107 cells/ml. After a 1-h incubation, Tran35S-label (ICN) was added to 20 μCi/ml, and the cells were labeled for 1 h. Cells were washed with water and resuspended in YE5S supplemented with 1 mg/ml cold methionine and 10 mm HU. At different time points, cells were harvested, washed with STOP buffer, and lysed in NETN buffer (20 mm Tris-HCl, pH 8.0, 100 mm NaCl, 1 mm EDTA, 0.5% Triton X-100) with protease inhibitors. 1 mg of the extract was incubated with glutathione-Sepharose beads for 2hat4 °C, and the bound Spd1-GST was washed three times with 1 ml of NETN buffer and then eluted with 100 mm glutathione in NETN. The eluted Spd1-GST was mixed with SDS loading buffer and resolved by SDS-PAGE. The gel was dried and the radioactive bands visualized by autoradiography and quantified by PhosphorImager. In Vivo Ubiquitination—Log phase cells expressing Spd1-GST along with a control untagged strain were grown in YE5S in the presence of 10 mm HU for 3 h. Cell extraction and GSH bead binding were performed exactly as for the pulse-chase assay except that 5 μm N-ethylmaleimide was added to the lysis buffer, and Spd1-GST was eluted with SDS-loading dye. 25 mg of total protein of the control and Spd1-GST strains and 4 mg of total protein for the Δddb1Spd1-GST strain were used to normalize the amount of Spd1-GST. The Spd1-GST-ubiquitin conjugates were visualized by Western blotting. Cds1 Kinase Assay—1-mg protein extracts were incubated with 0.5 μl of anti-Cds1 rabbit antibody prebound to 20 μl of protein G beads for 1 h at 4 °C with constant mixing. The kinase reaction was performed as described previously (6Bondar T. Mirkin E.V. Ucker D.S. Walden W.E. Mirkin S.M. Raychaudhuri P. J. Biol. Chem. 2003; 278: 37006-37014Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). FACS—Preparation of cell ghosts and flow cytometry were done as described previously (6Bondar T. Mirkin E.V. Ucker D.S. Walden W.E. Mirkin S.M. Raychaudhuri P. J. Biol. Chem. 2003; 278: 37006-37014Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). S Phase Proteolysis of Spd1 Requires Ddb1—We showed that the S. pombe strain lacking expression of ddb1 exhibited a defective S phase, including slow replication, which could be explained by the increased Cds1 kinase activity in that strain (6Bondar T. Mirkin E.V. Ucker D.S. Walden W.E. Mirkin S.M. Raychaudhuri P. J. Biol. Chem. 2003; 278: 37006-37014Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). Interestingly, the signalosome mutant Δcsn1, which failed to cause proteolysis of Spd1 in S phase, also exhibited an extended S phase and increased Cds1 activity (8Liu C. Powell K.A. Mundt K. Wu L. Carr A.M. Caspari T. Genes Dev. 2003; 9: 1130-1140Crossref Scopus (158) Google Scholar, 11Mundt K.E. Porte J. Murray J.M. Brikos C. Christensen P.U. Caspari T. Hagan I.M. Millar J.B. Simanis V. Hofmann K. Carr A.M. Curr. Biol. 1999; 9: 1427-1430Abstract Full Text Full Text PDF PubMed Google Scholar). Therefore, we investigated whether Ddb1 is required for the proteolysis of Spd1. The proteolysis of Spd1 is critical for the biosynthesis of dNTPs, which is essential for DNA synthesis. A deficiency in Spd1 proteolysis is expected to slow down S phase progression and cause replicative stress, which would explain many of the phenotypes (including the increased Cds1 activity) observed in the Δddb1 strain (6Bondar T. Mirkin E.V. Ucker D.S. Walden W.E. Mirkin S.M. Raychaudhuri P. J. Biol. Chem. 2003; 278: 37006-37014Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). We compared the steady-state levels of Spd1 in the Δddb1 strain with that in the wild type strain in Western blot assays using a specific antiserum raised against Spd1. The results of that analysis clearly indicated a significant accumulation of Spd1 in the Δddb1 strain compared with the wild type (Fig. 1A). The proteolysis of Spd1 occurs in S phase during each cycle of division to allow for an increase in the RNR activity (8Liu C. Powell K.A. Mundt K. Wu L. Carr A.M. Caspari T. Genes Dev. 2003; 9: 1130-1140Crossref Scopus (158) Google Scholar). To analyze the cell cycle proteolysis of Spd1, we compared the cdc25-22 strain with the Δddb1 cdc25-22 strain (19Russell P. Nurse P. Cell. 1986; 45: 145-153Abstract Full Text PDF PubMed Scopus (709) Google Scholar). The cdc25-22 encodes a temperature-sensitive allele of cdc25, which allows synchronization of the cells in G2 phase of the cell cycle. The cells were arrested by incubating for 3 h at a nonpermissive temperature (35 °C) followed by release to a permissive temperature (22 °C). At different times after the release, cells were harvested for protein extraction and cell cycle distribution analysis. The cell cycle distribution was determined by counting the septation index (Fig. 1B), which confirmed the synchronous progression of the cells through the cell cycle. Cell extracts were prepared following a procedure described previously (6Bondar T. Mirkin E.V. Ucker D.S. Walden W.E. Mirkin S.M. Raychaudhuri P. J. Biol. Chem. 2003; 278: 37006-37014Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). 0.5-mg extracts were subjected to Western blot analysis, which was probed with Spd1 antibody or with Cds1 antibody (as a loading control). The level of the Spd1 protein decreased in S phase of the wild type cells, but there was no detectable change in the level of Spd1 observed in the extracts from the Δddb1 strain (Fig. 1B). The lack of decrease in the levels of Spd1 in the Δddb1 strain is consistent with a requirement for Ddb1 in the proteolysis of Spd1 in S phase. Treatment with HU, which reduces the levels of dNTPs and causes an S phase arrest, was shown to cause proteolysis of Spd1 (8Liu C. Powell K.A. Mundt K. Wu L. Carr A.M. Caspari T. Genes Dev. 2003; 9: 1130-1140Crossref Scopus (158) Google Scholar). The proteolysis of Spd1 occurs in HU-treated cells because the cells are in S phase. Moreover, a proteolysis of Spd1 increases the activity of RNR which helps recovery of the cells from HU arrest. We measured the steady-state level of the Spd1 protein in the wild type strain and in the Δddb1 strain or in strains in which the endogenous Spd1 is replaced by Spd1-GST. The treatment with 10 mm HU for 3 h caused a dramatic reduction in the steady-state levels of native Spd1 or Spd1-GST in the wild type background. However, in the Δddb1 background, the levels of Spd1 (Fig. 2A) or Spd1-GST (Fig. 2B) remained essentially unaffected. To confirm further a deficiency in the proteolysis of Spd1 in the Δddb1 strain, we determined the decay rate of Spd1-GST in the wild type and in the Δddb1 cells after a treatment with HU. The cells were pulse labeled with [35S]methionine for 1 h before the addition of HU. The pulse-labeled cells were transferred to medium containing unlabeled methionine (chase) and 10 mm HU. At different time intervals, cells were harvested, and the cell lysates were bound to GSH beads to purify the Spd1-GST protein. After an extensive wash, the proteins bound to the GSH beads were eluted and subjected to SDS-PAGE followed by autoradiography (Fig. 3A). The band intensities were quantified by PhosphorImager and plotted against time of chase (Fig. 3B). As expected, the addition of HU caused a much faster decay of Spd1-GST in the wild type background relative to that observed in the Δddb1 strain treated with HU (Fig. 3B). The half-life of Spd1-GST in the Δddb1 strain was greater than 3 h in the presence of HU, whereas in the wild type background the half-life was less than 2 h. These observations further confirm the notion that the proteolysis of Spd1 is defective in the Δddb1 strain.Fig. 3Efficient decay and ubiquitination of Spd1 require Ddb1. A, Spd1-GST strains (wild type (wt) and Δddb1 background) were labeled with [35S]methionine, and the label was chased for 3 h in the presence of 10 mm HU. Extracts were bound to glutathione beads, eluted and resolved by SDS-PAGE, and visualized by autoradiography. The band intensities were quantified by PhosphorImager (B). C, Spd1-GST or Δddb1 Spd1-GST cells were treated with 10 mm HU for 3 h, and Spd1-GST was purified on glutathione beads and subjected to SDS-PAGE. The extract of an untagged strain was used as a control for nonspecific binding of ubiquitinated proteins to the glutathione beads. The ubiquitin-Spd1-GST conjugates were detected by Western blotting with anti-ubiquitin (α-Ub) antibody. The same membrane was reprobed with anti-GST antibody to verify equal loading of Spd1-GST protein.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Because it was shown that Spd1 is degraded through the ubiquitin-proteasome pathway (8Liu C. Powell K.A. Mundt K. Wu L. Carr A.M. Caspari T. Genes Dev. 2003; 9: 1130-1140Crossref Scopus (158) Google Scholar), we sought to investigate whether the Δddb1 strain is deficient in ubiquitinating Spd1-GST. To enrich the cell population for S phase cells where Spd1 is normally degraded, we looked at the ubiquitination of Spd1-GST in the presence of HU. The extracts from the HU-treated wild type and the Δddb1 strains expressing Spd1-GST were compared for the levels of ubiquitinated Spd1-GST. Because the level of Spd1-GST in the HU-treated Δddb1 strain is at least 6-fold higher than that in the wild type background, we normalized extract loads to the amounts of Spd1-GST. The extracts were bound to GSH beads to purify the Spd1-GST protein. Also, to control for the binding of nonspecific ubiquitinated protein to GSH beads, an equal amount of extract from an untagged wild type strain was used in a parallel set. The bound fractions were analyzed for ubiquitinated Spd1-GST using a Western blot assay and a monoclonal antibody against ubiquitin. The blot was also probed with GST antibody to confirm approximately equal loading of the Spd1-GST. As seen in Fig 3C, polyubiquitinated Spd1-GST was detected from cells with wild type background but not the Δddb1 background. The results are congruent with the notion that the Δddb1 strain is deficient in inducing the ubiquitination of Spd1. Deletion of the Spd1 Gene Reduces the Constitutively High Cds1 Activity in Δddb1 Strain—We showed that the Δddb1 strain exhibited a constitutively high basal activity of the replication checkpoint kinase Cds1 (6Bondar T. Mirkin E.V. Ucker D.S. Walden W.E. Mirkin S.M. Raychaudhuri P. J. Biol. Chem. 2003; 278: 37006-37014Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). The high basal Cds1 activity was linked to many if not all growth-related defects and DNA damage sensitivity in the Δddb1 strain because mutation in the cds1 gene reversed the defects, and the double mutant Δddb1 Δcds1 behaved like the wild type strain. It was, however, not clear why the Δddb1 strain exhibited a high basal level of the Cds1 activity. Interestingly, the Δcsn1 strain also exhibited a high basal Cds1 activity, and deletion of the spd1 gene reduced the Cds1 activity to a level observed for the wild type cells. Therefore, we sought to investigate whether a deletion of the spd1 gene would reduce the Cds1 activity in Δddb1 cells. We constructed a double mutant, Δddb1 Δspd1, lacking the expression of both ddb1 and spd1 genes. The double mutant was compared with the single mutants and with the wild type strain for the basal Csd1 activity. To assay for the Cds1 activity, cell extracts were immunoprecipitated with an antibody against Cds1. The immunoprecipitates were collected on protein A-Sepharose beads. The beads containing the immunopurified Cds1 were used to measure the activity of Cds1. In parallel, aliquots of the extracts were subjected to Western blot assays to compare the protein level of Cds1 from the various extracts (Fig. 4). The kinase activity of Cds1 was measured following a procedure described previously using myelin basic protein and [γ-32P]ATP as substrates. The reaction product was analyzed by SDS-PAGE followed by autoradiography. The phosphorylated MBP was quantified by PhosphorImager. Consistent with our previous results, the Δddb1 strain exhibited a significantly high level of the activated Cds1. In addition, we observed that the double mutant lacking expression of both Δddb1 and Δspd1 exhibited a much lower level of active Cds1. The level of active Cds1 in the double mutant was comparable with the Δspd1 single mutant strain (Fig. 4). These observations are consistent with the notion that the high Cds1 activity in Δddb1 strain is a result of the deficiency in the proteolysis of Spd1. Mutation in the spd1 Gene Reversed the Growth and Size Abnormalities in the Δddb1 Strain—The Δddb1 cells exhibited several growth-related defects, including elongated cell phenotype, increased doubling time, small colony size, and slow S phase progression. We attributed those defects in the Δddb1 cells to the high basal Cds1 activity. Because the spd1 mutation attenuated the Cds1 activity in the Δddb1 cells, we predicted that the growth-related defects in the Δddb1 strain would not be observed in the Δddb1 Δspd1 double mutant. The Δddb1 mutant displays slow growth, which is reflected by smaller colony size compared with the wild type cells. If Spd1 is the cause of t