Importin-β Mediates Cdc7 Nuclear Import by Binding to the Kinase Insert II Domain, Which Can Be Antagonized by Importin-α

We investigated the nuclear import mechanism of Cdc7, which is essential for the initiation of DNA replication. Here we report that importin-β binds directly to Cdc7 via the Kinase Insert II domain, promoting its nuclear import. Although both importin-α and -β bind to Cdc7 via the Kinase Insert II domain in a mutually independent manner, the binding affinity of Cdc7 for importin-β is ∼10 times higher than for importin-α at low protein concentrations of an equimolar ratio. Immunodepletion of importin-β, but not importin-α, abrogates Cdc7 nuclear import, and the addition of importin-β to the importin-depleted cytosol restores Cdc7 nuclear import. Furthermore, transduction of anti-importin-β, but not anti-importin-α antibodies, into live cells inhibits Cdc7 nuclear import. Unexpectedly, we found that Cdc7 nuclear import is inhibited by competitive binding of importin-α to Cdc7. Further studies by site-directed mutagenesis suggest that Lys306 and Lys309 within the Kinase Insert II domain are critical for Cdc7 nuclear localization. We investigated the nuclear import mechanism of Cdc7, which is essential for the initiation of DNA replication. Here we report that importin-β binds directly to Cdc7 via the Kinase Insert II domain, promoting its nuclear import. Although both importin-α and -β bind to Cdc7 via the Kinase Insert II domain in a mutually independent manner, the binding affinity of Cdc7 for importin-β is ∼10 times higher than for importin-α at low protein concentrations of an equimolar ratio. Immunodepletion of importin-β, but not importin-α, abrogates Cdc7 nuclear import, and the addition of importin-β to the importin-depleted cytosol restores Cdc7 nuclear import. Furthermore, transduction of anti-importin-β, but not anti-importin-α antibodies, into live cells inhibits Cdc7 nuclear import. Unexpectedly, we found that Cdc7 nuclear import is inhibited by competitive binding of importin-α to Cdc7. Further studies by site-directed mutagenesis suggest that Lys306 and Lys309 within the Kinase Insert II domain are critical for Cdc7 nuclear localization. The Cdc7-Dbf4 kinase functions as a molecular switch for the activation of individual origins of DNA replication (oris) throughout S phase (1Bousset K. Diffley J.F. Genes Dev. 1998; 12: 480-490Crossref PubMed Scopus (239) Google Scholar, 2Donaldson A.D. Fangman W.L. Brewer B.J. Genes Dev. 1998; 12: 491-501Crossref PubMed Scopus (182) Google Scholar, 3Jares P. Blow J.J. Genes Dev. 2000; 14: 1528-1540PubMed Google Scholar, 4Masai H. Sato N. Takeda T. Arai K. Front. Biosci. 1999; 4: D834-D840Crossref PubMed Google Scholar, 5Owens J.C. Detweiler C.S. Li J.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12521-12526Crossref PubMed Scopus (85) Google Scholar, 6Zou L. Stillman B. Mol. Cell. Biol. 2000; 20: 3086-3096Crossref PubMed Scopus (266) Google Scholar). To be functional, both the catalytic Cdc7 and regulatory Dbf4 subunits have to move into the nucleus, bind to chromatin, and associate with each other (7Jackson A.L. Pahl P.M. Harrison K. Rosamond J. Sclafani R.A. Mol. Cell. Biol. 1993; 13: 2899-2908Crossref PubMed Scopus (214) Google Scholar, 8Sclafani R.A. Jackson A.L. Mol. Microbiol. 1994; 11: 805-810Crossref PubMed Scopus (45) Google Scholar, 9Masai H. Arai K. J. Cell. Physiol. 2002; 190: 287-296Crossref PubMed Scopus (151) Google Scholar). Although Dbf4 contains a classical nuclear localization sequence (cNLS) 2The abbreviations used are: cNLS, classical nuclear localization sequence; CHO, Chinese hamster ovary; IBB domain, importin-β-binding domain; NPC, nuclear pore complex; PCNA, proliferating cell nuclear antigen; huCdc7, human Cdc7; GFP, green fluorescent protein; GST, glutathione S-transferase; FITC, fluorescein isothiocyanate; PBS, phosphate-buffered saline. 2The abbreviations used are: cNLS, classical nuclear localization sequence; CHO, Chinese hamster ovary; IBB domain, importin-β-binding domain; NPC, nuclear pore complex; PCNA, proliferating cell nuclear antigen; huCdc7, human Cdc7; GFP, green fluorescent protein; GST, glutathione S-transferase; FITC, fluorescein isothiocyanate; PBS, phosphate-buffered saline. (10Sato N. Sato M. Nakayama M. Saitoh R. Arai K. Masai H. Genes Cells. 2003; 8: 451-463Crossref PubMed Scopus (43) Google Scholar), Cdc7 does not have any known NLS. It was initially suggested that Dbf4 binds and transports Cdc7 into the nucleus (11Marx J. Science. 1995; 270: 1585-1587Crossref PubMed Scopus (14) Google Scholar). However, more recent data have shown that Cdc7 is located in the nucleus even when Dbf4 is not produced (12Guo B. Lee H. Somatic Cell Mol. Genet. 1999; 25: 159-171Crossref PubMed Scopus (6) Google Scholar, 13Guo B. Lee H. Gene (Amst.). 2001; 264: 249-256Crossref PubMed Scopus (7) Google Scholar, 14Weinreich M. Stillman B. EMBO J. 1999; 18: 5334-5346Crossref PubMed Scopus (227) Google Scholar, 15Jiang W. Hunter T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14320-14325Crossref PubMed Scopus (67) Google Scholar, 16Sato N. Arai K. Masai H. EMBO J. 1997; 16: 4340-4351Crossref PubMed Scopus (121) Google Scholar). The Cdc7 protein consists of 11 putative kinase domains that are highly conserved in all known Cdc7-related mammalian proteins (12Guo B. Lee H. Somatic Cell Mol. Genet. 1999; 25: 159-171Crossref PubMed Scopus (6) Google Scholar, 15Jiang W. Hunter T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14320-14325Crossref PubMed Scopus (67) Google Scholar, 16Sato N. Arai K. Masai H. EMBO J. 1997; 16: 4340-4351Crossref PubMed Scopus (121) Google Scholar). In addition, there are two kinase inserts: the Kinase Insert II spans from amino acids 203 to 370, and the Kinase Insert III from 440 to 538 (12Guo B. Lee H. Somatic Cell Mol. Genet. 1999; 25: 159-171Crossref PubMed Scopus (6) Google Scholar, 15Jiang W. Hunter T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14320-14325Crossref PubMed Scopus (67) Google Scholar, 16Sato N. Arai K. Masai H. EMBO J. 1997; 16: 4340-4351Crossref PubMed Scopus (121) Google Scholar, 17Kim J.M. Sato N. Yamada M. Arai K. Masai H. J. Biol. Chem. 1998; 273: 23248-23257Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). A potential short Kinase Insert I also exists at the amino acid residues 75-88. The amino acid sequences of the kinase inserts are the most diverse regions of the entire Cdc7 protein, and thus the inserts are thought to be involved in species-specific regulation and/or interactions with other proteins such as Dbf4 (4Masai H. Sato N. Takeda T. Arai K. Front. Biosci. 1999; 4: D834-D840Crossref PubMed Google Scholar). Proteins containing a cNLS are imported into the nucleus by an importin-α/-β heterodimer (18Gorlich D. Kutay U. Annu. Rev. Cell Dev. Biol. 1999; 15: 607-660Crossref PubMed Scopus (1667) Google Scholar, 19Jans D.A. Xiao C.Y. Lam M.H. Bioessays. 2000; 22: 532-544Crossref PubMed Scopus (476) Google Scholar, 20Mosammaparast N. Pemberton L.F. Trends Cell Biol. 2004; 14: 547-556Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar). In this transportation mode, the cNLS in a cargo protein is recognized and bound by importin-α through its carboxyl-terminal region, which, in turn, is bound by importin-β via its amino-terminal importin-β binding (IBB) domain. The cargo·importin-α/-β complex is transported into the nucleus through the nuclear pore complex (NPC) (18Gorlich D. Kutay U. Annu. Rev. Cell Dev. Biol. 1999; 15: 607-660Crossref PubMed Scopus (1667) Google Scholar, 20Mosammaparast N. Pemberton L.F. Trends Cell Biol. 2004; 14: 547-556Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar). Several recent data have demonstrated that certain proteins are transported into the nucleus by importin-α alone (21Kotera I. Sekimoto T. Miyamoto Y. Saiwaki T. Nagoshi E. Sakagami H. Kondo H. Yoneda Y. EMBO J. 2005; 24: 942-951Crossref PubMed Scopus (78) Google Scholar), or by importin-β alone (22Moore J.D. Yang J. Truant R. Kornbluth S. J. Cell Biol. 1999; 144: 213-224Crossref PubMed Scopus (174) Google Scholar, 23Nagoshi E. Imamoto N. Sato R. Yoneda Y. Mol. Biol. Cell. 1999; 10: 2221-2233Crossref PubMed Scopus (103) Google Scholar, 24Truant R. Cullen B.R. Mol. Cell. Biol. 1999; 19: 1210-1217Crossref PubMed Google Scholar, 25Xiao Z. Liu X. Lodish H.F. J. Biol. Chem. 2000; 275: 23425-23428Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 26Forwood J.K. Lam M.H. Jans D.A. Biochemistry. 2001; 40: 5208-5217Crossref PubMed Scopus (83) Google Scholar, 27Yamasaki H. Sekimoto T. Ohkubo T. Douchi T. Nagata Y. Ozawa M. Yoneda Y. Genes Cells. 2005; 10: 455-464Crossref PubMed Scopus (66) Google Scholar). We examined the regulatory mechanism of human Cdc7 (huCdc7) nuclear transportation using in vitro and in vivo assays. We found that huCdc7 is directly bound and translocated into the nucleus by importin-β. The binding site is mapped to the Cdc7 Kinase Insert II, and the Lys306 and Lys309 residues within this domain are critical for Cdc7 nuclear localization. Most interestingly, importin-α can competitively bind to the Cdc7 Kinase Insert II, and can thus effectively inhibit importin-β-mediated Cdc7 nuclear transportation. Our data also raises the possibility that the binding of Cdc7 to importin-α could be involved in the activation or maintenance of the replication checkpoint in response to cell damaging agents such as irradiation and anticancer agents. Cell Culture and DNA Transfection—HeLa and HEK293 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum (HyClone, Logan, UT) and a combination of penicillin (50 units/ml) and streptomycin (50 μg/ml). Chinese hamster ovary (CHO) cells were grown in minimal essential medium supplemented with 10% Fetal Clone II (HyClone). Cells grown on a coverglass were transfected with plasmids for 12 h using Lipofectamine PLUS™ reagent as suggested by the supplier (Invitrogen) and as described previously (28Guo B. Pearce A.G. Traulsen K.E. Rintala A.C. Lee H. BioTechniques. 2001; PubMed Scopus Google Scholar). and plasmids were by a the entire human Cdc7 into the site of or into the site of protein in a Cdc7 was into the site of Cdc7 were by DNA into or The plasmids and were a of M. for H. P. A. T. D. J. Cell Biol. 2000; PubMed Scopus Google Scholar). The was a of S. A. Kornbluth (22Moore J.D. Yang J. Truant R. Kornbluth S. J. Cell Biol. 1999; 144: 213-224Crossref PubMed Scopus (174) Google Scholar). The plasmids and were by D. of D. S. R. R.A. E. S. Biol. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar, U. E. D. EMBO J. 1997; 16: PubMed Scopus Google Scholar). was by the DNA from to of into a amino acids was by a of into a and were from or the and proliferating cell nuclear and all were from were from Cell and were from and GST, and proteins were in and were by affinity on as suggested by the were using with or proteins were using affinity to the and as described previously D. S. R. R.A. E. S. Biol. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar, U. E. D. EMBO J. 1997; 16: PubMed Scopus Google Scholar). to nuclear import all proteins were and transfected with were at h with containing 10% Z. Liu X. Lodish H.F. J. Biol. Chem. 2000; 275: 23425-23428Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). The cell were for h at with with GST, or To the proteins with Cdc7, cells were with containing and concentrations of cell were with a protein and were with as The were with and the proteins bound to the were by to which was to the to The proteins were using a by with containing 10% and in or proteins were for at with the with proteins were a using a and the was for at The was for h with containing and by with or importin-β proteins at The was times with for and proteins bound to the were by was using the grown on a coverglass were with for at and were with in for with containing bovine serum for h at cells were with for h at The cells were times with each and with or for at cells were with PBS, on a and by was used to the In import in cells were as described previously L. J. Cell Biol. PubMed Scopus Google with cells grown on a coverglass were with containing for The cells were with and was the cells on a were with a of nuclear and at for A nuclear cell cytosol and an of and L. J. Cell Biol. PubMed Scopus Google Scholar). import was by using the To and/or importin-β, cell cytosol was with of and/or of at for which was for h with The bound to were by at for was times to and/or -β and/or importin-β were to the cytosol of the the importin-β was used as a of import of cell as previously described D. S. R. R.A. E. S. Biol. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar, J. Cell Biol. PubMed Scopus Google Scholar, E. L. J. J. Cell. 1999; PubMed Scopus Google Scholar). and HEK293 cells grown on a coverglass were with or using a to the was used as 12 h Cdc7 was by with an and -β with to the nuclear localization of Cdc7 proteins at the of this Cdc7 proteins were to be used for this protein was in the nucleus To huCdc7 is transported into the nucleus, we the proteins with Cdc7 by a using with or as described and of the proteins bound to the that and proteins with Cdc7, PCNA, and not data not huCdc7 does not a cNLS, the binding of huCdc7 with was The data shown in could not and -β bind to Cdc7 in a mutually or a classical cargo complex studies that the is The binding of to Cdc7 is with the data that protein is the of Cdc7 N. Arai K. Masai H. EMBO J. 1997; 16: 4340-4351Crossref PubMed Scopus (121) Google Scholar, M. W. S. A. K. Y. A. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar, W. D. Hunter T. EMBO J. 1999; 18: PubMed Scopus Google PCNA, a nuclear protein, and a protein, not with Cdc7, that proteins not with Cdc7 at all for than for at the classical importin-α/-β Cdc7 nuclear import, we investigated the interactions Cdc7 and -β using an in vitro GST, or on was with cell from cells GFP, Cdc7, or shown in using or that both Cdc7 and with and which is with the data in with containing a cNLS from but not with a cNLS that the and of proteins not the interactions Sekimoto T. Imamoto N. K. T. Yoneda Y. EMBO J. 1997; 16: PubMed Scopus Google previously that or proteins not the of the of this is that PCNA, which does not a cNLS, be imported into the nucleus by directly with importin-β. However, we not the nuclear transportation mechanism To the of interactions Cdc7 and we with shown in Cdc7 directly with independent of the other The using proteins also a the data in to suggest that Cdc7 higher affinity for importin-β than for and we a more by and of and each on a The was with by with an shown in and the binding affinity of Cdc7 for both and -β is However, the affinity of Cdc7 for importin-β is higher than for at low protein the of Cdc7 bound to importin-β at the of was that of at the was and Furthermore, of was to the of the that the of protein contains as than importin-β is at the of and -β the binding affinity of Cdc7 for importin-β is higher than for protein concentrations the data shown in and suggest that Cdc7 binding affinity for importin-β in the or of a low of the possibility that importin-β is for Cdc7 nuclear import. but Cdc7 the that importin-β is directly for Cdc7 nuclear import, we a of in vitro nuclear import in the or of importin-β as described shown in cell cytosol can effectively Cdc7 in the that the cell cytosol contains all the for Cdc7 nuclear import. To for Cdc7 nuclear we an in vitro nuclear import using cytosol of both and cytosol not Cdc7 nuclear import I and Cdc7 nuclear localization was when both and -β proteins were to the cytosol III and of to the cytosol not Cdc7 nuclear import and Cdc7 nuclear import was when importin-β alone was to the cytosol and importin-β is directly for the Cdc7 nuclear transportation. To this we alone from the with the data shown in the or of not Cdc7 nuclear import In the cytosol importin-β could not Cdc7 nuclear which was when importin-β was that importin-β, but not is for Cdc7 nuclear import, both can bind to that the of the proteins was by To importin-β also Cdc7 nuclear import in we or into cells using a transduction The transduction of and into cells not the nuclear localization of Cdc7 In the transduction of into cells effectively inhibited Cdc7 nuclear import that importin-β is for Cdc7 nuclear import in We also a using HEK293 and found the as shown in not on the data from in vitro and in vivo we that importin-β, but not importin-α, directly Cdc7 nuclear import. is by the data by The Kinase Insert II for the of Cdc7 to and to the mechanism of importin-β Cdc7 nuclear import. To this we Cdc7 data not and examined of binding to importin-β. shown in binding affinity for both and there is binding site within the amino acids at the In Kinase Insert II and Cdc7 binding affinity for both and that the amino acid residues Kinase Insert II is for Cdc7 binding to both and The or alone bound to importin-β, at a However, could bind to To the in vitro binding of Cdc7 to importin-β is directly to Cdc7 nuclear we in vitro assays. cells were with or Cdc7 in the of and an that cytosol was not used in this protein and were not the cargo protein was not to be in the nucleus but at the by this (18Gorlich D. Kutay U. Annu. Rev. Cell Dev. Biol. 1999; 15: 607-660Crossref PubMed Scopus (1667) Google Scholar, 20Mosammaparast N. Pemberton L.F. Trends Cell Biol. 2004; 14: 547-556Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar). with in vitro binding Cdc7 and at the in the of importin-β, but and The and also at the NPC, at that in vitro binding of importin-β to Cdc7 is directly to Cdc7 nuclear transportation in an Further suggested that the Cdc7 amino acids is for Cdc7 nuclear localization not we and within this region, and examined the nuclear localization of each in transfected shown in Cdc7 and were to the However, the Cdc7 was in that the Lys306 and Lys309 the residues are critical for huCdc7 nuclear localization. the binding affinity of importin-β for the Cdc7 is times than for Cdc7 that importin-β directly bind to Cdc7 and into the with for to Cdc7 and Cdc7 both and -β bind to the of the Cdc7 protein, with importin-β for binding to Cdc7. We a binding for Cdc7 and importin-β in the of concentrations of protein a of and Cdc7 bound to importin-β, but not to the of and -β was to both of bound to Cdc7 Further in to a inhibited the binding Cdc7 and importin-β The data shown in also an that and -β can bind to Cdc7 in the of the other and The binding of Cdc7 to and -β raises the possibility that inhibit the importin-β-mediated Cdc7 nuclear in to its classical of promoting of the cargo·importin-α/-β complex into the The of complex by could be of a of with Cdc7 through the Kinase Insert II domain as by the data shown in could the complex by binding to which of is we a binding using the IBB domain a of the of with that the of Cdc7 binding to importin-β is to the interactions of to Cdc7 through the Kinase Insert II domain and To the binding of Cdc7 to and was using a The of proteins and in was a and by The protein in each were by using or with the data shown in the of and proteins with that Cdc7 a complex with importin-β, but not with and all raises the possibility that certain all Cdc7, and a of and can effectively with importin-β for Cdc7, we the is also with the that the molecular of protein in the be but not and Cdc7 and that Cdc7 can a complex with which is with the data shown in We could with importin-β-mediated Cdc7 at the shown in can inhibit importin-β-mediated Cdc7 to the at a of and the at the data are with the that the of the complex by is directly to the of Cdc7 nuclear import. To the of on Cdc7 nuclear we nuclear import using cell is imported into the nucleus in the of the cytosol and an in the and a low of was to the not inhibit Cdc7 nuclear import and the of was to Cdc7 nuclear import was inhibited and the of was more in the cells in A and The for this and cells are We were by the that huCdc7 could bind to both importin-α and huCdc7 does not any known However, studies by and in vitro binding using of importin-α and -β to that huCdc7, bound by both a higher affinity for importin-β than for importin-α at low protein that a binding of Cdc7 to importin-β in the the low protein concentrations used for are more to be the Cdc7 can be transported into the nucleus of importin-α in the with this we found that the binding of Cdc7 by importin-β is directly to Cdc7 nuclear import. We demonstrated that the cytosol of both importin-α and -β not Cdc7 nuclear which was by importin-α and -β of importin-β alone to the cytosol of both importin-α and -β could Cdc7 nuclear import. In of importin-α to the cytosol not Cdc7 nuclear import. data that importin-β, but not is for Cdc7 nuclear import. We the using the importin-α or -β of importin-β, but not importin-α, inhibited Cdc7 nuclear and the cytosol with importin-β Cdc7 nuclear import. data that is the for the initiation of huCdc7 nuclear import, which was also with the of in vivo transduction and in vitro and In vitro binding using Cdc7 suggest that the Cdc7 Kinase Insert II acids contains binding for both importin-α and -β The and Cdc7 a binding affinity for importin-β, the not bind to importin-β at that the Kinase Insert II domain is and for binding to importin-α and both and could bind to importin-β, at but not to importin-α data raises the there are two for importin-β but site for importin-α within the Kinase Insert II domain; or there is binding site for both importin-α and -β within the entire Kinase Insert II the is of the two importin-β binding be at amino acids and the other at from the of protein in importin-β bind to two with there is binding site within the Kinase Insert II domain, both and regions of the amino acid In this each of the and protein alone have binding affinity for importin-β, both protein be for more binding to importin-β. the site is more than binding be within the Kinase Insert II domain. this we we the site a within a protein could inhibit Cdc7 nuclear localization as that the Kinase Insert II amino the of this of protein for binding is from the nuclear transportation. known proteins transported into the nucleus by importin-β to a of protein E. Imamoto N. Sato R. Yoneda Y. Mol. Biol. Cell. 1999; 10: 2221-2233Crossref PubMed Scopus (103) Google Scholar, 27Yamasaki H. Sekimoto T. Ohkubo T. Douchi T. Nagata Y. Ozawa M. Yoneda Y. Genes Cells. 2005; 10: 455-464Crossref PubMed Scopus (66) Google Scholar, Sekimoto T. E. Nagoshi E. A. Imamoto N. M. H. T. Yoneda Y. Science. 2003; PubMed Scopus Google Scholar). suggest that a binding is for importin-β-mediated protein nuclear transportation the binding of importin-β to a cargo protein is by found that the Lys306 and Lys309 residues are essential for huCdc7 nuclear localization as the Cdc7 was not in the data suggest that the of the Cdc7 that can with importin-β. importin-β-mediated protein nuclear localization also a amino acid sequence addition to the In this be of the potential of is from of in an importin-β-mediated protein nuclear localization when more proteins directly transported by importin-β are and The most in this is that importin-α can competitively the complex of Cdc7 with importin-β, which is directly to the of Cdc7 nuclear localization and importin-α inhibits of Cdc7 at the in the of the Cdc7 nuclear transportation by importin-α is at the in the to transportation through the Furthermore, this of Cdc7 nuclear import by importin-α is independent of importin-α binding to importin-β Our data from and suggest that the affinity Cdc7 and importin-β is when equimolar concentrations of importin-α and -β are in the of the complex in Cdc7 nuclear import. Cdc7 nuclear the two residues at and are by promoting binding Cdc7 and importin-β as the Cdc7 binding affinity for importin-β The binding Cdc7 and importin-α in not the equimolar importin-α Cdc7 nuclear transportation the of importin-α binding to Cdc7 also the potential of importin-α with importin-β of the the IBB of importin-α a E. M. L. J. Cell. 1998; 94: Full Text Full Text PDF PubMed Scopus Google Scholar, D.A. Trends Cell Biol. 2004; 14: Full Text Full Text PDF PubMed Scopus Google Scholar). a cargo protein contains a cNLS, this binding can is importin-α can bind to the cargo protein, by which the binding potential of importin-α to -β can also through the of the IBB domain from its E. M. L. J. Cell. 1998; 94: Full Text Full Text PDF PubMed Scopus Google Scholar, D.A. Trends Cell Biol. 2004; 14: Full Text Full Text PDF PubMed Scopus Google Scholar). It is known that cells can a replication checkpoint in response to cell damaging agents such as irradiation and anticancer agents H. J.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: PubMed Scopus Google Scholar, J.M. Lee H. Mol. Cell. Biol. 1994; 14: PubMed Scopus Google Scholar). Miyamoto Y. Saiwaki T. J. Y. I. S. M. Y. T. Yoneda Y. J. Cell Biol. 2004; PubMed Scopus Google have found that by irradiation can the nuclear of importin-α and inhibit a nuclear import. In an possibility that importin-α be to bind to Cdc7 in the nucleus importin-β is and is thus involved in the of replication initiation when a cell a an checkpoint can cell the Cdc7 binding to importin-α in the nucleus in response to cell have to in the of We are to D. and S. A. Kornbluth for and -β to M. for and R. for this to for in for and and for Cdc7 with

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