Degradation of Cdt1 during S Phase Is Skp2-independent and Is Required for Efficient Progression of Mammalian Cells through S Phase

Previous reports have shown that the N terminus of Cdt1 is required for its degradation during S phase (Li, X., Zhao, Q., Liao, R., Sun, P., and Wu, X. (2003) J. Biol. Chem. 278, 30854–30858; Nishitani, H., Lygerou, Z., and Nishimoto, T. (2004) J. Biol. Chem. 279, 30807–30816). The stabilization was attributed to deletion of the cyclin binding motif (Cy motif), which is required for its phosphorylation by cyclin-dependent kinases. Phosphorylated Cdt1 is subsequently recognized by the F-box protein Skp2 and targeted for proteasomal mediated degradation. Using phosphopeptide mapping and mutagenesis studies, we found that threonine 29 within the N terminus of Cdt1 is phosphorylated by Cdk2 and required for interaction with Skp2. However, threonine 29 and the Cy motif are not necessary for proteolysis of Cdt1 during S phase. Mutants of Cdt1 that do not stably associate with Skp2 or cyclins are still degraded in S phase to the same extent as wild type Cdt1, indicating that other determinants within the N terminus of Cdt1 are required for degrading Cdt1. We localized the region necessary for Cdt1 degradation to the first 32 residues. Overexpression of stable forms of Cdt1 significantly delayed entry into and completion of S phase, suggesting that failure to degrade Cdt1 prevents normal progression through S phase. In contrast, Cdt1 mutants that fail to interact with Skp2 and cyclins progress through S phase with similar kinetics as wild type Cdt1 but stimulate the re-replication caused by overexpressing Cdt1. Therefore, a Skp2-independent pathway that requires the N-terminal 32 residues of Cdt1 is critical for the degradation of Cdt1 in S phase, and this degradation is necessary for the optimum progression of cells through S phase. Previous reports have shown that the N terminus of Cdt1 is required for its degradation during S phase (Li, X., Zhao, Q., Liao, R., Sun, P., and Wu, X. (2003) J. Biol. Chem. 278, 30854–30858; Nishitani, H., Lygerou, Z., and Nishimoto, T. (2004) J. Biol. Chem. 279, 30807–30816). The stabilization was attributed to deletion of the cyclin binding motif (Cy motif), which is required for its phosphorylation by cyclin-dependent kinases. Phosphorylated Cdt1 is subsequently recognized by the F-box protein Skp2 and targeted for proteasomal mediated degradation. Using phosphopeptide mapping and mutagenesis studies, we found that threonine 29 within the N terminus of Cdt1 is phosphorylated by Cdk2 and required for interaction with Skp2. However, threonine 29 and the Cy motif are not necessary for proteolysis of Cdt1 during S phase. Mutants of Cdt1 that do not stably associate with Skp2 or cyclins are still degraded in S phase to the same extent as wild type Cdt1, indicating that other determinants within the N terminus of Cdt1 are required for degrading Cdt1. We localized the region necessary for Cdt1 degradation to the first 32 residues. Overexpression of stable forms of Cdt1 significantly delayed entry into and completion of S phase, suggesting that failure to degrade Cdt1 prevents normal progression through S phase. In contrast, Cdt1 mutants that fail to interact with Skp2 and cyclins progress through S phase with similar kinetics as wild type Cdt1 but stimulate the re-replication caused by overexpressing Cdt1. Therefore, a Skp2-independent pathway that requires the N-terminal 32 residues of Cdt1 is critical for the degradation of Cdt1 in S phase, and this degradation is necessary for the optimum progression of cells through S phase. During G1 phase of the cell cycle, large multi-protein complexes referred to as pre-replicative complexes (pre-RCs) 1The abbreviations used are: pre-RC, pre-replicative complex; CDK, cyclin-dependent kinase; Cy motif, cyclin binding motif; FACS, fluorescence-activated cell sorter; GST, glutathione S-transferase; HA, hemagglutinin; GFP, green fluorescent protein; ORC, origin recognition complex; WT, wild type. 1The abbreviations used are: pre-RC, pre-replicative complex; CDK, cyclin-dependent kinase; Cy motif, cyclin binding motif; FACS, fluorescence-activated cell sorter; GST, glutathione S-transferase; HA, hemagglutinin; GFP, green fluorescent protein; ORC, origin recognition complex; WT, wild type. assemble at replication origins throughout the genome, “licensing” the DNA for replication in S phase (1Blow J.J. Hodgson B. Trends Cell Biol. 2002; 12: 72-78Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar, 2Nishitani H. Lygerou Z. Genes Cells. 2002; 7: 523-534Crossref PubMed Scopus (219) Google Scholar). Pre-RCs form in a stepwise fashion, beginning with the six-subunit origin recognition complex (ORC) that binds to origins and recruits Cdc6 and Cdt1, which are, in turn, both required for the subsequent recruitment of the replicative helicase Mcm2-7 (3Bell S.P. Dutta A. Annu. Rev. Biochem. 2002; 71: 333-374Crossref PubMed Scopus (1392) Google Scholar). Transition to S phase is accompanied by a dramatic increase in the levels of cyclin-dependent kinases (Cdks), which activate pre-RCs by mediating recruitment of the replicative polymerases that begin DNA synthesis. High Cdk activity during S phase also inhibits the formation of new pre-RCs. Therefore, origins are prevented from becoming relicensed until the next M/G1 transition, at which point cyclins are rapidly degraded and pre-RCs are again allowed to assemble. Consistent with this model, perturbation of Cdk levels such as inhibiting Cdk activity in G2 is sufficient to stimulate re-replication (4Broek D. Bartlett R. Crawford K. Nurse P. Nature. 1991; 349: 388-393Crossref PubMed Scopus (289) Google Scholar, 5Dahmann C. Diffley J.F. Nasmyth K.A. Curr. Biol. 1995; 5: 1257-1269Abstract Full Text Full Text PDF PubMed Scopus (307) Google Scholar, 6Itzhaki J.E. Gilbert C.S. Porter A.C. Nat. Genet. 1997; 15: 258-265Crossref PubMed Scopus (138) Google Scholar). In addition to Cdk activity, metazoans have developed another mechanism for preventing re-replication involving the protein geminin. Geminin prevents inappropriate licensing by binding to and inhibiting Cdt1 (7Tada S. Li A. Maiorano D. Mechali M. Blow J.J. Nat. Cell Biol. 2001; 3: 107-113Crossref PubMed Scopus (390) Google Scholar, 8Wohlschlegel 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, 9Lee C. Hong B. Choi J.M. Kim Y. Watanabe S. Ishimi Y. Enomoto T. Tada S. Cho Y. Nature. 2004; 430: 913-917Crossref PubMed Scopus (118) Google Scholar). Cdt1 overexpression or geminin silencing results in re-replication and genomic instability, highlighting the importance of properly regulating Cdt1 (10Vaziri 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 (337) Google Scholar, 11Zhu W. Chen Y. Dutta A. Mol. Cell. Biol. 2004; 24: 7140-7150Crossref PubMed Scopus (204) Google Scholar, 12Melixetian M. Ballabeni A. Masiero L. Gasparini P. Zamponi R. Bartek J. Lukas J. Helin K. J. Cell Biol. 2004; 165: 473-482Crossref PubMed Scopus (215) Google Scholar). The levels of Cdt1 are regulated during the cell cycle, being stable in G1 when pre-RCs are formed and degraded in S phase when pre-RCs are fired and disassembled (8Wohlschlegel 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, 13Nishitani H. Taraviras S. Lygerou Z. Nishimoto T. J. Biol. Chem. 2001; 276: 44905-44911Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar). Regulation is primarily at the protein level, as mRNA levels of Cdt1 remain constant throughout the cell cycle (13Nishitani H. Taraviras S. Lygerou Z. Nishimoto T. J. Biol. Chem. 2001; 276: 44905-44911Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar). It was recently shown that Cdt1 is phosphorylated in vivo and that its phosphorylation is required for interaction with the F-box protein Skp2 (14Li 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). Skp2 is a component of the SCF complex, which functions as an E3 ubiquitin ligase. The SCFSkp2 complex has been implicated in the ubiquitination and ensuing proteasomal degradation of several cell cycle proteins at the G1/S transition, with the Skp2 subunit specifically recognizing phosphorylated substrates. Subsequently, it was shown that Cdt1 is phosphorylated by Cdks, suggesting that Cdt1 degradation is mediated through Cdk phosphorylation, which targets it for ubiquitination by SCFSkp2 (15Liu Li X. Zhao Q. Wu X. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, N. Y. T. A. T. H. M. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, H. Lygerou Z. Nishimoto T. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). However, for this mechanism is still cells have been to in the of Cdt1 protein with that of wild type cells K. H. S. N. S. M. S. T. Cell. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). silencing of Skp2 by Cdt1 levels (14Li 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 Skp2 levels also has been shown to entry into S phase by H. R. K. D. Cell. 1995; Full Text PDF PubMed Scopus Google Scholar, N. L. J. Genes 2004; PubMed Scopus Google Scholar, A.C. A. M. Nat. Cell Biol. PubMed Scopus Google Scholar). Cdt1 is stable in G1 phase, cell cycle progression in Cdt1 Skp2 degradation in a mechanism for degrading Cdt1 origin Walter J.C. Genes PubMed Scopus Google Scholar). we to the of the Cdt1, and Cdk phosphorylation in cells by the Cdt1 required for Skp2 point mutants of Cdt1, we the by Skp2 we that the N terminus of Cdt1 is required for its mutants of Cdt1 that do not interact with Skp2 and cyclins are still degraded during S phase, suggesting the of Skp2-independent for Cdt1 in S phase. mutants stimulate the re-replication by overexpressing Cdt1, suggesting that phosphorylation by interaction with Skp2 has a Cdt1 activity of In contrast, of Cdt1 mutants that are stable during S phase a delayed entry into and completion of S phase, suggesting that failure to degrade Cdt1 prevents progression through S phase. Cell and and cells as in with or for to the a green fluorescent was at a and by with to cells in G1 phase. S phase cells from into and for of cyclin in (8Wohlschlegel 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, J.A. Dwyer B.T. Dutta A. Mol. Cell. Biol. 2001; PubMed Scopus Google Scholar). for Cdt1 by Cdt1 from into P. Dutta A. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google the and Cdt1 in Cdt1 was by with and into the and of in Cdt1 the with the and for and for and and for N-terminal mutants of Cdt1 by to the in Cdt1 and into the and of was by the and and into the of by Cdt1 and cyclin from as (8Wohlschlegel 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, J.A. Dwyer B.T. Dutta A. Mol. Cell. Biol. 2001; PubMed Scopus Google for in in in and with of and by the addition of of in a of at for by the addition of by in vivo phosphopeptide cells with and with and with Cdt1 was by binding to was with and by and with The to Cdt1 was from the and to an by by the at and cell by cells in and with was a of for to as A. M. Zamponi R. Masiero L. Helin K. J. 2004; PubMed Scopus Google Scholar). The used Cdt1 (8Wohlschlegel J.A. Dwyer B.T. Dhar S.K. Cvetic C. Walter J.C. Dutta A. Science. 2000; 290: 2309-2312Crossref PubMed Scopus (584) Google cyclin P. Dutta A. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Skp2 M. Walter J.C. J. 2004; PubMed Scopus Google geminin (8Wohlschlegel J.A. Dwyer B.T. Dhar S.K. Cvetic C. Walter J.C. Dutta A. Science. 2000; 290: 2309-2312Crossref PubMed Scopus (584) Google and and to with with and with and for at cells a and The N of Cdt1 during S the N terminus of Cdt1 are several phosphorylation as as the cyclin binding motif (Cy at residues (15Liu Li X. Zhao Q. Wu X. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, N. Y. T. A. T. H. M. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). Therefore, we an N-terminal of Cdt1 by the first We also to an in to properly to the H. Lygerou Z. Nishimoto T. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). cells with Cdt1 and and in with cells in cells from in S phase by cells from into and with used to the cell cycle phase. that Cdt1 is stable in G1 and degraded in S phase, to is with Cdt1 results also that we are not the by overexpressing Cdt1. In contrast, is stable in S phase Therefore, the first of Cdt1 are necessary for its degradation during S phase. that the N terminus of Cdt1 is not required for its licensing indicating that the N terminus of Cdt1 not in a and protein A. Blow J.J. J. 24: PubMed Scopus Google Scholar, A. Li A. M. Blow J.J. PubMed Scopus Google Scholar). the of Cdt1 in and in phosphorylation of Cdt1 is required for with Skp2 N. Y. T. A. T. H. M. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google we the phosphorylation of Cdt1 in and in Cdt1 was phosphorylated in cyclin Cdt1 was by and to by mapping phosphorylated residues within the first threonine 29 and are by a with the Cdk phosphorylation The threonine is within a Cdk that an of the in phosphorylation are phosphorylated in we cells with a the N terminus of Cdt1, with for and results are shown in and that threonine 29 and are also phosphorylated in are other phosphorylation in that not the in vivo results the in phosphorylation and a point for mutagenesis 29 for Cdt1 with the phosphopeptide mapping we point mutants that threonine 29 to or to The Cy motif was by residues which to of the Cy motif, to has been shown to the stable of cyclins with Cdt1 in and in vivo N. Y. T. A. T. H. M. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). We also a into cells in to interaction with Skp2 by type Cdt1 Skp2 However, of the Cy motif the stable interaction Cdt1 and Skp2 as (15Liu Li X. Zhao Q. Wu X. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, N. Y. T. A. T. H. M. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). to Skp2 In contrast, of to Skp2 The also not Skp2 as Therefore, we that threonine 29 and the Cy motif are both necessary for Cdt1 interaction with Skp2. The of threonine 29 is similar to that of Cdk that the Cy motif to the Cdk J.A. Dutta A. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). The threonine 29 and the Cy motif is also the of residues required for recognition by cyclin J.A. Dutta A. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). Cdt1 Mutants with Skp2 and during S and fail to interact with Skp2 in vivo and Skp2 has been to the mechanism by which Cdt1 is degraded during the cell cycle (14Li 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 we and to to degradation. or Cdt1 into cells and at G1 or S phase a and as in was to the point mutants and a stable form of Cdt1 within the same with cyclin to G1 and S phase of the cell is stable in S phase and degraded in S phase similar to Cdt1 and of Cdt1 was still as of cells with the to and during S phase caused by of the cell cycle, we also the of the Cdt1 proteins in an S phase by cells at a from Cdt1 and still degraded as cells S phase was stable interaction with Skp2 is not required for the degradation of Cdt1 during S phase, suggesting the of Skp2-independent for degrading Cdt1 during S phase. of Cdt1 for S of is stable in S phase, we that another within the first that targets Cdt1 for degradation of Skp2 and cyclin Therefore, we mutants of the N terminus of Cdt1 that the first and mutants during the cell cycle, was and stable during S phase and The addition of during S phase also not have an the levels of and we also Cdt1 with an deletion of residues was still degraded in S phase suggesting that in the N terminus of Cdt1 that Cdt1 degradation during S phase. Therefore, we that residues are necessary for degradation of Cdt1 during S phase. to Cdt1 in an S forms of Cdt1 that stable in S phase, we the of Cdt1 degradation during the cell cycle and cells with Cdt1 at from cells by FACS, a was and cells The cell cycle of cells wild type Cdt1, or to However, the cell cycle progression of cells with or was significantly and cells G1 and a to the other Cdt1 and However, at and cells with and DNA of G1 phase, the of cells with wild type Cdt1, or S phase and DNA when of the wild type and cells S phase, cells or still cells with DNA The of stable Cdt1 to a delayed entry into S phase a cell cycle the cells DNA The of S phase is also with and The point at which S phase cells are for cells and cells wild type Cdt1 is the and the of cells indicating that it to However, cells which begin to S phase at the are still in S phase at the Therefore, Cdt1 also the of S phase. The of was to suggesting that the region of Cdt1 for the with is within the first 32 mutants progression through S phase. cells the Cdt1 a and at and and to as 29 and Cy motif mutants stimulate cells the Cdt1 from a the of cells DNA was for cells with Cdt1 the and of from an for for with the the stable forms of Cdt1 as we the of replication The of Cdt1 is the of the Mcm2-7 complex Mcm2-7 replication in the of Cdt1. cell and from cells wild type Cdt1, or an at during the cell that both wild type Cdt1 and with the with wild type Cdt1 being degraded at the point to S phase. was with from and as cells S phase. Therefore, with was by the of wild type Cdt1 or suggesting that at origins to the same extent as in that the complexes we also the of with requires Mcm2-7 and is an of Cdt1 wild type Cdt1 and of at the point similar to that Cdt1 Mcm2-7 complexes to S phase. Therefore, we the that stable forms of Cdt1 are in a of wild type Cdt1 caused the of cells with DNA to re-replication as (10Vaziri 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 (337) Google Scholar, M. 2004; PubMed Scopus Google Scholar). progression through S phase was by overexpressing and overexpression increase the of cells with DNA from to and suggesting that Skp2 and Cdk phosphorylation the of Cdt1 to are not to stabilization of Cdt1 as that are at similar levels In contrast, and in cells primarily DNA with a of cells with DNA as with wild type Cdt1 in addition to S phase stable forms of Cdt1 a to stimulate However, the in re-replication to the with progression through S phase as it was that Cdt1 with Skp2 in a (14Li 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, Li X. Zhao Q. Wu X. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, N. Y. T. A. T. H. M. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). that this is mediated by phosphorylation of threonine 29 is phosphorylated by Cdk complexes in and was found to phosphorylated in of threonine 29 or the Cy motif binding of Cdt1 to Skp2 in vivo Therefore, binding to cyclin is to with but of threonine 29 also by being phosphorylated by Cdt1 that associate with cyclins is to Skp2 with a threonine another for threonine a Cy motif to associate with Skp2. cyclin binding and phosphorylation of threonine 29 are The importance of is with of of which a phosphorylated We the that another that threonine threonine 29 is within a Cdk that another is it another Cdk or a protein the kinases to In the of a Cy motif in the phosphorylation of a Cdk by a Cdk is of Cy and Cdk phosphorylation in another replication Cdc6 J.A. Dutta A. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). In that a Cy motif at residues to the Cdk significantly the of a for phosphorylation by both cyclin and cyclin we that a Cy interaction with cyclin the phosphorylation of threonine 29 of Cdt1 by cyclin phosphorylation is necessary for Cdt1 to interact with Skp2. mutants of Cdt1 that do not interact with Skp2 or cyclins are degraded during S phase similar to wild type Cdt1 protein and not the that Skp2 is in degrading Cdt1, but it that are Skp2-independent that degrade Cdt1 during S phase. Cdk phosphorylation still required for degrading Cdt1, but the stable of Cdt1 with cyclins is not mechanism the N-terminal 32 residues of Cdt1, as deletion of this region Cdt1 during S phase. stabilization of Cdt1 was Skp2 which that Skp2 is for Cdt1 degradation (14Li 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). silencing of Skp2 has been shown to targets N. L. J. Genes 2004; PubMed Scopus Google Scholar, A.C. A. M. Nat. Cell Biol. PubMed Scopus Google Scholar, H. A. C. M. W. Nat. Cell Biol. PubMed Scopus Google in G1 at the during the cell cycle when Cdt1 is we used Cdt1 point cell cycle caused by Skp2 levels do not reports in other are also with In Cdk phosphorylation not Cdt1 during S phase M. 2004; PubMed Scopus Google Scholar). stabilization of Cdt1 required the cell cycle in G1 by overexpressing or In Cdt1 degradation origin was to Walter J.C. Genes PubMed Scopus Google Scholar). to degradation of Cdt1 in S phase also not to interaction with Skp2 or It is that of threonine 29 or the Cy motif increase the re-replication caused by overexpression of Cdt1 the of the Cdt1 protein Therefore, Cdk phosphorylation in regulating Cdt1 of degradation. of Cdt1 has been shown to its to DNA N. Y. T. A. T. H. M. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). such as and Cdc6 are also regulated by phosphorylation of proteolysis A. Mol. Cell. Biol. 2004; 24: PubMed Scopus Google Scholar, S.P. Genes 2004; PubMed Scopus Google Scholar, C. Li J.J. Nature. 2001; PubMed Scopus Google Scholar, Mol. Cell. Biol. 2004; 24: PubMed Scopus Google Scholar). Therefore, it is that the interaction of Cdt1 with or with Cdc6 or its in Mcm2-7 proteins is regulated by phosphorylation threonine Consistent with the that Cdt1 proteolysis is mediated through Skp2-independent the cell cycle progression of cells cyclin and Skp2 binding mutants that of wild type Cdt1 type Cdt1, and progress through S phase with the same kinetics as cells and However, cells a significantly delayed entry into and completion of S phase, suggesting that failure to degrade Cdt1 prevents the progression through S phase. the Cy motif Skp2 binding the of the N terminus for Cdt1 deletion of the first 32 residues of Cdt1 was sufficient to degradation of Cdt1 during S phase. also the same cell cycle as the that the with are to its It is failure to degrade Cdt1 prevents S phase is that the stable forms of Cdt1 as that with the normal of Cdt1. The of Cdt1 is is subsequent such as recruitment of to do not the of Cdt1. both and in cells a stable form of Cdt1, we that the S phase progression is not to of wild type Cdt1 stable forms of Cdt1 stimulate which also a We that the of Cdt1 during S phase. activate that inappropriate formation and by or the cell degradation of Cdt1 required for origin by a the N terminus of Cdt1 a that DNA synthesis. cells overexpressing wild type Cdt1 through S phase similar to re-replication are with that overexpression of Cdt1 re-replication and preventing entry into (10Vaziri 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 (337) Google Scholar, 11Zhu W. Chen Y. Dutta A. Mol. Cell. Biol. 2004; 24: 7140-7150Crossref PubMed Scopus (204) Google Scholar, 12Melixetian M. Ballabeni A. Masiero L. Gasparini P. Zamponi R. Bartek J. Lukas J. Helin K. J. Cell Biol. 2004; 165: 473-482Crossref PubMed Scopus (215) Google Scholar). Skp2 and cyclin binding mutants of Cdt1 the re-replication with wild type Cdt1 of with Cdt1, of Cdk phosphorylation in re-replication when M. 2004; PubMed Scopus Google Scholar). it was shown that of Cdk activity during was sufficient to of Cdt1 A. M. Zamponi R. Masiero L. Helin K. J. 2004; PubMed Scopus Google Scholar). Therefore, it is that Cdk phosphorylation is during a in phase for preventing Cdt1 and origin geminin is the of Cdt1 during the of the cell that Cdt1 do not the re-replication with wild type Cdt1. to the S phase, which entry into S phase progression by Cdt1 is that origins to of Cdt1 geminin has been shown to Cdt1 to Cdt1 and is Cdt1 is degraded Walter J.C. Genes PubMed Scopus Google Scholar, D. W. Mechali M. Cell 2004; PubMed Scopus Google Scholar). Cdt1 geminin at origins and prevents It to stable Cdt1 mutants that fail to interact with geminin to In we the importance of the N terminus of Cdt1 for its degradation during S phase. However, the N terminus of Cdt1 is required for both cyclin and Skp2 we that is required for degrading Cdt1 during S phase. Therefore, Cdt1 proteolysis during S phase is mediated Skp2-independent In the of the degradation to the first 32 of Cdt1, we that overexpression of stable forms of Cdt1 results in a delayed entry into and progression through S phase. In contrast, Cdt1 mutants that fail to interact with Skp2 and cyclins stimulate the re-replication when overexpressing Cdt1, do not Cdt1 in S phase. is it that phosphorylation by Cdk2 and interaction with Skp2 have a Cdt1 of it for degradation. in the and into the in for regulating Cdt1. In Cdt1 overexpression has been shown to have P. J. J.M. Choi K. 2002; PubMed Google suggesting that Cdt1 to the of Therefore, the in regulating Cdt1 by which cells to re-replication and genomic