An Evolutionarily Conserved Function of Proliferating Cell Nuclear Antigen for Cdt1 Degradation by the Cul4-Ddb1 Ubiquitin Ligase in Response to DNA Damage

Abstract

The DNA replication licensing factor Cdt1 is degraded by the ubiquitin-proteasome pathway during S phase of the cell cycle, to ensure one round of DNA replication during each cell division and in response to DNA damage to halt DNA replication. Constitutive expression of Cdt1 causes DNA re-replication and is associated with the development of a subset of human non-small cell-lung carcinomas. In mammalian cells, DNA damage-induced Cdt1 degradation is catalyzed by the Cul4-Ddb1-Roc1 E3 ubiquitin ligase. We report here that overexpression of the proliferating cell nuclear antigen (PCNA) inhibitory domain from the CDK inhibitors p21 and p57, but not the CDK-cyclin inhibitory domain, blocked Cdt1 degradation in cultured mammalian cells after UV irradiation. In vivo soluble Cdt1 and PCNA co-elute by gel filtration and associate with each other physically. Silencing PCNA in cultured mammalian cells or repression of pcn1 expression in fission yeast blocked Cdt1 degradation in response to DNA damage. Unexpectedly, deletion of Ddb1 in fission yeast cells also accumulated Cdt1 in the absence of DNA damage. We suggest that the Cul4-Ddb1 ligase evolved to ubiquitinate Cdt1 during normal cell growth as well as in response to DNA damage and a separate E3 ligase, possibly SCFSkp2, evolved to either share or take over the function of Cdt1 ubiquitination during normal cell growth and that PCNA is involved in mediating Cdt1 degradation by the Cul4-Ddb1 ligase in response to DNA damage. The DNA replication licensing factor Cdt1 is degraded by the ubiquitin-proteasome pathway during S phase of the cell cycle, to ensure one round of DNA replication during each cell division and in response to DNA damage to halt DNA replication. Constitutive expression of Cdt1 causes DNA re-replication and is associated with the development of a subset of human non-small cell-lung carcinomas. In mammalian cells, DNA damage-induced Cdt1 degradation is catalyzed by the Cul4-Ddb1-Roc1 E3 ubiquitin ligase. We report here that overexpression of the proliferating cell nuclear antigen (PCNA) inhibitory domain from the CDK inhibitors p21 and p57, but not the CDK-cyclin inhibitory domain, blocked Cdt1 degradation in cultured mammalian cells after UV irradiation. In vivo soluble Cdt1 and PCNA co-elute by gel filtration and associate with each other physically. Silencing PCNA in cultured mammalian cells or repression of pcn1 expression in fission yeast blocked Cdt1 degradation in response to DNA damage. Unexpectedly, deletion of Ddb1 in fission yeast cells also accumulated Cdt1 in the absence of DNA damage. We suggest that the Cul4-Ddb1 ligase evolved to ubiquitinate Cdt1 during normal cell growth as well as in response to DNA damage and a separate E3 ligase, possibly SCFSkp2, evolved to either share or take over the function of Cdt1 ubiquitination during normal cell growth and that PCNA is involved in mediating Cdt1 degradation by the Cul4-Ddb1 ligase in response to DNA damage. Cdt1, first identified in the fission yeast Schizosaccharomyces pombe as a G1 START component Cdc10-dependent transcript whose loss-of-function prevents DNA replication (1.Hofmann J.F. Beach D. EMBO J. 1994; 13: 425-434Crossref PubMed Scopus (182) Google Scholar), binds to the origin recognition complex with Cdc6 at the origin of replication and together with Cdc6 and origin recognition complex recruits the minichromosome maintenance 2–7 (MCM2–7) to assemble the prereplication complexes during G1, thereby controlling the initiation of DNA replication (2.Bell S.P. Dutta A. Annu. Rev. Biochem. 2002; 71: 333-374Crossref PubMed Scopus (1392) Google Scholar). Constitutive expression of Cdt1 alone in Caenorhabditis elegans, with Cdc6 in S. pombe or with Cdc6 and cyclin A-cdk2 in p53-deficient mammalian cells causes DNA re-replication (3.Zhong W. Feng H. Santiago F.E. Kipreos E.T. Nature. 2003; 423: 885-889Crossref PubMed Scopus (259) Google Scholar, 4.Yanow S.K. Lygerou Z. Nurse P. EMBO J. 2001; 20: 4648-4656Crossref PubMed Scopus (77) Google Scholar, 5.Vaziri C. Saxena S. Jeon Y. Lee C. Murata K. Machida Y. Wagle N. Hwang D.S. Dutta A. Mol. Cell. 2003; 11: 997-1008Abstract Full Text Full Text PDF PubMed Scopus (338) Google Scholar). Constitutive expression of Cdt1 is also associated with the development of a subset of human non-small lung carcinomas (6.Karakaidos P. Taraviras S. Vassiliou L.V. Zacharatos P. Kastrinakis N.G. Kougiou D. Kouloukoussa M. Nishitani H. Papavassiliou A.G. Lygerou Z. Gorgoulis V.G. Am. J. Pathol. 2004; 165: 1351-1365Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar), indicating the critical importance of regulating Cdt1 level for both initiating DNA replication and maintaining genome integrity. In addition to Cdc10-dependent transcriptional regulation, at least four mechanisms have been proposed for controlling Cdt1 function at the protein level; Cdt1 is exported from the nucleus in S phase in the budding yeast Saccharomyces cerevisiae (7.Tanaka S. Diffley J.F. Nat. Cell Biol. 2002; 4: 198-207Crossref PubMed Scopus (217) Google Scholar), is inhibited by the binding of geminin from S to M phase in metazoans (8.Wohlschlegel J.A. Dwyer B.T. Dhar S.K. Cvetic C. Walter J.C. Dutta A. Science. 2000; 290: 2309-2312Crossref PubMed Scopus (584) Google Scholar), is degraded in human cell lines, possibly by the SCFSkp2 E3 2The abbreviations used are: E3, ubiquitin-protein isopeptide ligase; PCNA, proliferating cell nuclear antigen; siRNA, small interfering RNA; HU, hydroxyurea; MMS, methylmethane sulfonate.2The abbreviations used are: E3, ubiquitin-protein isopeptide ligase; PCNA, proliferating cell nuclear antigen; siRNA, small interfering RNA; HU, hydroxyurea; MMS, methylmethane sulfonate. ubiquitin ligase (9.Nishitani H. Lygerou Z. Nishimoto T. Nurse P. Nature. 2000; 404: 625-628Crossref PubMed Scopus (368) Google Scholar, 10.Li X. Zhao Q. Liao R. Sun P. Wu X. J. Biol. Chem. 2003; 278: 30854-30858Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar), and is degraded in response to DNA damage and during normal C. elegans embryogenesis by the Cul4-Ddb1-Roc1 ubiquitin E3 ligase (3.Zhong W. Feng H. Santiago F.E. Kipreos E.T. Nature. 2003; 423: 885-889Crossref PubMed Scopus (259) Google Scholar, 11.Higa L.A. Mihaylov I.S. Banks D.P. Zheng J. Zhang H. Nat. Cell Biol. 2003; 5: 1008-1015Crossref PubMed Scopus (256) Google Scholar, 12.Hu J. McCall C.M. Ohta T. Xiong Y. Nat. Cell Biol. 2004; 6: 1003-1009Crossref PubMed Scopus (294) Google Scholar). Evolving multiple distinct mechanisms to negatively regulate the level of Cdt1 protein presumably functions to meet the needs of stopping DNA replication irreversibly at multiple stages of the cell cycle and development and rapidly in response to genotoxic insults. The mechanisms underlying the ubiquitin-mediated degradation of Cdt1 remain incompletely understood. In particular, the identity and components of the Cdt1 E3 ubiquitin ligase are currently confusing. Two different E3 ligases, a CUL1-dependent SCFSkp2 E3 (10.Li X. Zhao Q. Liao R. Sun P. Wu X. J. Biol. Chem. 2003; 278: 30854-30858Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 13.Sugimoto N. Tatsumi Y. Tsurumi T. Matsukage A. Kiyono T. Nishitani H. Fujita M. J. Biol. Chem. 2004; 279: 19691-19697Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar) and a Cul4-Ddb1-dependent E3 (3.Zhong W. Feng H. Santiago F.E. Kipreos E.T. Nature. 2003; 423: 885-889Crossref PubMed Scopus (259) Google Scholar, 11.Higa L.A. Mihaylov I.S. Banks D.P. Zheng J. Zhang H. Nat. Cell Biol. 2003; 5: 1008-1015Crossref PubMed Scopus (256) Google Scholar, 12.Hu J. McCall C.M. Ohta T. Xiong Y. Nat. Cell Biol. 2004; 6: 1003-1009Crossref PubMed Scopus (294) Google Scholar), have been linked to Cdt1 degradation. Critically missing from the current understanding of these two ligases in promoting Cdt1 ubiquitination is the signal(s) that recruits Cdt1 to either ligase. We previously reported that Ddb1 preferentially associated with a slow migrating form of Cdt1, which likely corresponds to phosphorylated Cdt1 (12.Hu J. McCall C.M. Ohta T. Xiong Y. Nat. Cell Biol. 2004; 6: 1003-1009Crossref PubMed Scopus (294) Google Scholar). The nature of this DNA damage induced Cdt1 kinase and whether Cdt1 phosphorylation is required for binding with Ddb1 are yet to be determined. Separately, a cyclin A-dependent CDK has been reported to promote Cdt1 phosphorylation and subsequent binding with the F-box protein Skp2 (13.Sugimoto N. Tatsumi Y. Tsurumi T. Matsukage A. Kiyono T. Nishitani H. Fujita M. J. Biol. Chem. 2004; 279: 19691-19697Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 14.Liu E. Li X. Yan F. Zhao Q. Wu X. J. Biol. Chem. 2004; 279: 17283-17288Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar), resulting in Cdt1 degradation during the normal cell cycle. Co-immunoprecipitation analysis showed that cells treated with the proteasome inhibitor MG132 prior to UV irradiation had increased Skp2-Cdt1 association (15.Kondo T. Kobayashi M. Tanaka J. Yokoyama A. Suzuki S. Kato N. Onozawa M. Chiba K. Hashino S. Imamura M. Minami Y. Minamino N. Asaka M. J. Biol. Chem. 2004; 279: 27315-27319Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar), raising the possibility that both SCFSkp2 and Cul4-Ddb1 could mediate Cdt1 degradation following UV-induced DNA damage. In an effort to clarify the role of CDKs in regulating Cdt1 ubiquitination, we examined how CDK inhibitors may affect Cdt1 degradation during the DNA damage response. This investigation did not identify any evidence for the requirement of a CDK in regulating Cdt1 degradation in response to DNA damage but instead led to the unexpected finding that the proliferating cell nuclear antigen (PCNA) is required for Cdt1 degradation. Plasmids, Cell Culture, and Cell Transfection—Plasmids expressing human p21, p21ΔPCNA (M148A/F151A), p27, p57QT(residues 142–198), and p57QTΔPCNA (L271A/F275A) were described previously (16.Watanabe H. Pan Z.-Q. Schreiber-Agus N. DePinho R.A. Hurwitz J. Xiong Y. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1392-1397Crossref PubMed Scopus (144) Google Scholar), and the procedures for immunoprecipitation and immunoblotting were as described previously (12.Hu J. McCall C.M. Ohta T. Xiong Y. Nat. Cell Biol. 2004; 6: 1003-1009Crossref PubMed Scopus (294) Google Scholar). All human cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum in a 37 °C incubator with 5% CO2. Cell transfections were performed using a calcium phosphate buffer. Antibodies, Proteins, Immunological Procedures, Mass Spectrometric Analysis, and Size Exclusion Chromatography—Antibodies to hemagglutinin (HA) (12CA5; Roche Applied Science, Mannheim, Germany), to protein A (Sigma), to PCNA (PC10, Santa Cruz Biotechnology, Santa Cruz, CA), to actin (NeoMarkers, Fremont, CA), and to p21, p27, and p57 (16.Watanabe H. Pan Z.-Q. Schreiber-Agus N. DePinho R.A. Hurwitz J. Xiong Y. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1392-1397Crossref PubMed Scopus (144) Google Scholar, 17.Franklin D.S. Xiong Y. Mol. Biol. Cell. 1996; 7: 1587-1599Crossref PubMed Scopus (105) Google Scholar) were either purchased commercially or described previously. Rabbit polyclonal antibodies to Cul4A, Ddb1, and Cdt1 have been described previously (12.Hu J. McCall C.M. Ohta T. Xiong Y. Nat. Cell Biol. 2004; 6: 1003-1009Crossref PubMed Scopus (294) Google Scholar). To purify the Cdt1 complex, 10 150-mm plates of HEK 293T cells were transfected with pcDNA3-myc3-Cdt1 and lysed with a 0.5% Nonidet P-40 lysis buffer (50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 0.5% Nonidet P-40, 50 mm NaF). Pooled lysates were incubated with affinity purified anti-Cdt1 (10 μg) antibody. Immunocomplexes were precipitated by protein A-agarose beads and then eluted by incubating with a molar excess of antigen peptide. Eluted immunocomplexes were resolved on an SDS-PAGE gel, stained with Coomassie Blue, and protein bands digested with trypsin before mass spectrometric analysis at the University of North Carolina-Duke Proteomics Core Facility. For size exclusion chromatography, HeLa cells were lysed in 0.5% Nonidet P-40 lysis buffer (50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 0.5% Nonidet P-40, 50 mm NaF) and fractionated on a Sepharose 200 gel-filtration column. The fractions were resovled by SDS-PAGE before the levels of Cdt1 and PCNA were determined by immunoblotting. RNA Interference—All siRNA oligonucleotides were synthesized with 3′-NN overhangs by Dharmacon (Lafayette, CO) in a purified and annealed duplex form. The sequences targeting each human gene were as follows: Ddb1, 5′-CCUGUUGAUUGCCAAAAAC-3′ and PCNA, 5′-UCAAGGACCUCAUCAACGA-3′. Opti-MEM medium (500 μl) was mixed with Lipofectamine 2000 reagent (10 μl) for 5 min and then incubated with 10 μl (20 mm) of siRNA for 20 min at room temperature. Dulbecco's modified Eagle's, 10% fetal bovine serum medium (1.5 ml) was added to the mixture, and the entire 2.5 ml was added to HeLa cells cultured on a 60-mm plate at 30–40% confluence. S. pombe Genetics and Strain Construction—Strain construction, gene deletion, and epitope tagging were performed by standard genetic techniques (18.Bahler J. Wu J.Q. Longtine M.S. Shah N.G. McKenzie III, A. Steever A.B. Wach A. Philippsen P. Pringle J.R. Yeast. 1998; 14: 943-951Crossref PubMed Scopus (1756) Google Scholar). Nmt81-Pcn1 was a gift from Dr. Teresa Wang of Stanford University (19.Arroyo M.P. Wang T.S. Methods. 1999; 18: 335-348Crossref PubMed Scopus (4) Google Scholar). To determine how CDK activity may affect Cdt1 degradation in response to DNA damage, we overexpressed the CDK inhibitors p21 and p27 in HEK 293T cells and determined the steady-state levels of Cdt1 protein following UV irradiation. Overexpression of p21 considerably inhibited Cdt1 degradation following UV irradiation (Fig. 1A). Although this finding is with the that CDK may Cdt1 phosphorylation and thereby promote degradation in response to DNA damage, overexpression of p27 had on Cdt1 degradation in the p21 and p27 have CDK in The these two CDK inhibitors in p21 a PCNA binding and inhibitory domain in the of p27 in regulating the phosphorylation of p27 and but has PCNA binding This to determine which in p21, the CDK-cyclin binding or PCNA binding is required for Cdt1 degradation following UV irradiation. The PCNA binding domain in p21 has been and required for the binding of p21 to PCNA have been identified J. Dutta A. Nature. PubMed Scopus Google Scholar, Y. Hurwitz J. J. Nature. PubMed Scopus Google Scholar, M. J.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, E. D.P. Biol. 5: Full Text Full Text PDF PubMed Scopus Google Scholar). of and to in p21 to as binding with PCNA M. J.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, E. D.P. Biol. 5: Full Text Full Text PDF PubMed Scopus Google Scholar) and to Cdt1 degradation following UV irradiation (Fig. The CDK inhibitor also a domain, to as the domain, that with the PCNA binding domain of p21 and binds with and the function of PCNA (16.Watanabe H. Pan Z.-Q. Schreiber-Agus N. DePinho R.A. Hurwitz J. Xiong Y. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1392-1397Crossref PubMed Scopus (144) Google Scholar). To the role of PCNA in regulating Cdt1 we the domain and examined Cdt1 degradation following UV irradiation. Overexpression of the but not a to as that with PCNA (16.Watanabe H. Pan Z.-Q. Schreiber-Agus N. DePinho R.A. Hurwitz J. Xiong Y. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1392-1397Crossref PubMed Scopus (144) Google Scholar), blocked Cdt1 degradation after UV irradiation (Fig. that of PCNA blocked Cdt1 degradation in response to DNA damage. also that CDKs that are inhibited by p21 and p27 are to be required for DNA damage-induced Cdt1 degradation. In the absence of DNA damage, overexpression of both p21 and p27 increased the steady-state level of Cdt1 the migrating form (Fig. 1A). This that either a is involved in regulating Cdt1 during normal cell or the Cdt1 from the G1 cell cycle by p21 or p27 subsequent was on the of during the DNA damage response. To evidence the function of PCNA in controlling Cdt1 we examined the of these two by gel Size exclusion that soluble PCNA and Cdt1 in a at mass of that is with the in vivo of a complex containing PCNA, Cdt1 (Fig. Cul4A, on the other is in mass fractions and is not in the mass fractions that both Cdt1 and The of of Cdt1 and PCNA and of from are with the possibility that PCNA is involved in promoting Cdt1 ubiquitination and subsequent degradation of Cdt1 by the ligase. a Cdt1 (12.Hu J. McCall C.M. Ohta T. Xiong Y. Nat. Cell Biol. 2004; 6: 1003-1009Crossref PubMed Scopus (294) Google Scholar), we Cdt1 complexes from an Nonidet and examined by Coomassie (Fig. were identified as the that were by a molar excess of antigen peptide. In addition to a that was identified as Cdt1 the were bands at a mass of Mass in addition to the Cdt1, two geminin and PCNA (Fig. The of geminin and PCNA from the of Cdt1 and these two Overexpression of p21 the association (Fig. that one by which p21 Cdt1 is to This needs to be as the possibility that p21 could also association by To in vivo evidence a role of PCNA in Cdt1 we first performed in cultured mammalian cells to determine how the expression of PCNA the steady-state level of Cdt1 with previously (12.Hu J. McCall C.M. Ohta T. Xiong Y. Nat. Cell Biol. 2004; 6: 1003-1009Crossref PubMed Scopus (294) Google Scholar), Ddb1 by siRNA accumulated Cdt1 in HeLa cells (Fig. of the steady-state level of PCNA by siRNA accumulated Cdt1 following UV irradiation as well (Fig. and but did not Cdt1 protein level in cells and The of PCNA on degradation of Cdt1 is likely to of PCNA For in vivo we fission which have PCNA, Cdt1, and the Cul4-Ddb1 E3 ligase. We fission yeast expressing Cdt1 the of in both and The steady-state level of is in in cells (Fig. and This the first evidence that in fission yeast Ddb1 is also involved in controlling Cdt1 degradation as in mammalian cells and that a E3 ligase in fission yeast cells also Cdt1 degradation during normal cell growth in the absence of DNA damage. We then examined the level of Cdt1 in and cells either of DNA replication or DNA damage. Unexpectedly, DNA damage of DNA replication by of cells with the steady-state level of (Fig. and To this we yeast expressing the of in both and is an inhibitor of and is also for degradation by the Cul4-Ddb1 ligase both during S phase and in response to DNA damage in S. pombe C. K. Wu T. 2003; PubMed Scopus Google Scholar, T. A. P. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar, C. C. R. PubMed Scopus Google Scholar). with of DNA replication degradation of and deletion of Ddb1 blocked degradation (Fig. and We also that during the normal cell cycle, the steady-state level of is in cells in cells (Fig. and that Ddb1 in fission yeast cells is involved in degradation of both during normal cell cycle and DNA replication is and the DNA replication a Cdt1 and degradation during the normal cell cycle. We that during S and Cdt1 are degraded before and after replication and an may to the ubiquitination of these two by the E3 ligase. We that in cells, Cdt1 accumulated to a level and of or and in and of indicating that Ddb1 is required for the degradation of both the level of these two is by an We that may a to the expression of but not Cdt1, in cells during each cycle. a Cdt1 to a level not cell to DNA replication and of cells with methylmethane a DNA damage Cdt1 degradation in cells (Fig. and or 5 and but had on the steady-state level of Cdt1 in cells and indicating that DNA Cdt1 degradation had evolved as as in yeast To whether PCNA is required for Cdt1 degradation in response to DNA damage in S. we a in which PCNA gene expression is the of the and is after the Cdt1 gene of expression by addition was by with an to PCNA (Fig. and and and of PCNA had on the steady-state level of in the absence of DNA damage and but blocked the degradation of Cdt1 in response to DNA damage and PCNA is required for Cdt1 degradation in response to DNA damage in S. Cdt1 was accumulated in cells in This is likely of PCNA the cell cycle and prevents Cdt1 Ddb1 cells to and In this we separate of evidence that a role of PCNA in mediating Cdt1 degradation by Cul4-Ddb1 in response to DNA damage. overexpression of the PCNA inhibitory domain of either p21 or p57, but not the CDK-cyclin inhibitory domain, blocked Cdt1 degradation in soluble PCNA and Cdt1 in the fractions and PCNA and Cdt1 associate with each other in PCNA in cultured mammalian cells and repression of PCNA expression in fission yeast both Cdt1 degradation following DNA damage. with the that Ddb1 is required for DNA damage-induced Cdt1 degradation in mammalian cells (12.Hu J. McCall C.M. Ohta T. Xiong Y. Nat. Cell Biol. 2004; 6: 1003-1009Crossref PubMed Scopus (294) Google Scholar) and in fission yeast (Fig. we suggest that Cdt1 degradation by Cul4-Ddb1 ligase in response to DNA damage evolved and has been during also is the role of PCNA in promoting Cdt1 We previously reported that Ddb1 to Cdt1 (12.Hu J. McCall C.M. Ohta T. Xiong Y. Nat. Cell Biol. 2004; 6: 1003-1009Crossref PubMed Scopus (294) Google Scholar), that PCNA is not required for Cdt1 to the Cul4-Ddb1 ligase. in Cdt1 ubiquitination has identified a of PCNA a role of PCNA in Cdt1 ubiquitination by the Cul4-Ddb1 ligase. factor missing from these is In particular, that four Cdt1, PCNA, Ddb1, and are associated with is likely that Cdt1 ubiquitination may in at the of DNA on and that PCNA a critical role in in Cdt1 ubiquitination, a function that is not to be in the current in or by In in Cdt1 is degraded during the of DNA replication on Walter J.C. PubMed Scopus Google Scholar). this Cdt1 degradation on is catalyzed by the Cul4-Ddb1 ligase or by CDK to be determined. The the role of Ddb1, a factor that was identified as a component of a DNA binding activity and has been associated with DNA during normal cell growth in the absence of DNA damage. deletion of Ddb1 in fission yeast cells accumulated Cdt1 as well as in cells (Fig. a function of Ddb1 in controlling normal cell to be determined whether this function of Ddb1 is also in to this is the role of Skp2 in Cdt1 degradation. evidence has that E3 ligase, possibly SCFSkp2, is involved in Cdt1 ubiquitination and degradation in mammalian cells (10.Li X. Zhao Q. Liao R. Sun P. Wu X. J. Biol. Chem. 2003; 278: 30854-30858Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 13.Sugimoto N. Tatsumi Y. Tsurumi T. Matsukage A. Kiyono T. Nishitani H. Fujita M. J. Biol. Chem. 2004; 279: 19691-19697Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 14.Liu E. Li X. Yan F. Zhao Q. Wu X. J. Biol. Chem. 2004; 279: 17283-17288Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). with the of multiple E3 for Cdt1 Ddb1, Cdt1 following DNA damage, was not to Cdt1 during normal cell growth L.A. Mihaylov I.S. Banks D.P. Zheng J. Zhang H. Nat. Cell Biol. 2003; 5: 1008-1015Crossref PubMed Scopus (256) Google Scholar, 12.Hu J. McCall C.M. Ohta T. Xiong Y. Nat. Cell Biol. 2004; 6: 1003-1009Crossref PubMed Scopus (294) Google Scholar). We have evidence in fission yeast that suggest the of E3 ligase in promoting Cdt1 of Ddb1 accumulated Cdt1 to a level (Fig. that E3 ligase in fission yeast that Cdt1 ubiquitination, activity be in with that of We suggest that the Cul4-Ddb1 ligase evolved to ubiquitinate Cdt1 both during normal cell growth as well as in response to DNA damage. in a separate E3 ligase evolved to either share or take over the function of Cdt1 ubiquitination during normal cell but DNA damage-induced Cdt1 degradation to be by the Cul4-Ddb1 ligase. We Dr. Teresa Wang for S. pombe McCall for the and other of the Xiong for this