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J. Biol. Chem., Vol. 282, Issue 41, 30029-30038, October 12, 2007

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Identification and Characterization of Human Cdc7 Nuclear Retention and Export Sequences in the Context of Chromatin Binding^*

Byung Ju Kim¹, So-Young Kim, and Hoyun Lee^¶2

From the Department of Biochemistry, Microbiology and Immunology, the Faculty of Medicine, University of Ottawa, Ottawa, Ontario K1M 8M5, Canada, the Tumour Biology Group, Northeastern Ontario Regional Cancer Program at the Sudbury Regional Hospital, Sudbury, Ontario P3E 5J1, Canada, and the ^¶Department of Medical Sciences, The Northern Ontario School of Medicine, Sudbury, Ontario P3E 2C6, Canada

The Cdc7 serine/threonine kinase activates the initiation of DNA replication by phosphorylating MCM proteins that are bound to the origins of DNA replication. We reported previously that human Cdc7 nuclear import is mediated directly by importin- through its binding to the Cdc7 nuclear localization sequence (NLS). Here, we report that human Cdc7 nuclear localization is regulated by two additional elements: nuclear retention (NRS) and export sequences (NES). Cdc7 proteins imported into the nucleus are retained in the nucleus by associating with chromatin, for which NRS-(306–326) is essential. Importantly, this binding appears to be specific to the origin of DNA replication, because the binding of wild-type Cdc7 to origin is 2.4-fold higher than to non-origin DNA. Furthermore, an NRS-defective Cdc7 mutant could not be retained in the nucleus, although it was imported into the nucleus normally. Together, our data suggest that NRS plays an important role in the activation of DNA replication by Cdc7. The Cdc7 proteins unassociated with chromatin are bound by CRM1 via two NES elements: NES1 at 458–467 within kinase insert III, and NES2 at 545–554 within the kinase IX domain. The primary function of the Cdc7-CRM1 association may be to translocate nuclear Cdc7 to the cytoplasm. However, the binding of CRM1 with Cdc7 at NES2 raises an interesting possibility that CRM1 may also down-regulate Cdc7 by masking its kinase domain.

Received for publication, May 4, 2007 , and in revised form, August 16, 2007.

^* This work was supported by grants from the Canadian Institutes of Health Research (MOP79473) and the Natural Sciences and Engineering Research Council of Canada (203528-02) (to H. L.). This report is a part of the PhD thesis of B. J. K. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S4.

¹ Supported in part by an Ontario Graduate Scholarship and the University of Ottawa Excellence Award.

² To whom correspondence should be addressed: Tumour Biology Group, Northeastern Ontario Regional Cancer Program at the Sudbury Regional Hospital. Tel.: 705-522-6237, ext. 2703; E-mail, hlee{at}hrsrh.on.ca.

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