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

Byung Ju Kim,So-Young Kim,Hoyun Lee

doi:10.1074/jbc.m703705200

Byung Ju Kim, So-Young Kim + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m703705200

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Abstract

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-beta 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.

Highlights

Human Cdc7 protein comprises 11 kinase domains and two kinase inserts [17, 18]
To further dissect Cdc7 nuclear localization sequence (NLS), we generated and charand resuspended in DNase-free water, which was subjected to acterized several mutant constructs bearing a series of deletions quantification by real-time PCR (7900HT, Applied Biosystems) within the Kinase Insert II domain (Figs. 1A and supplemental using SYBR green master mix (Applied Biosystems)
The Regulation of Cdc7 Nuclear Import and Retention—We previously reported that human Cdc7 is imported into the nucleus by importin-␤, for which the NLS at the Cdc7 Kinase Insert II domain is essential [19]

Summary

Introduction

Human Cdc7 (huCdc7) protein comprises 11 kinase domains and two kinase inserts [17, 18]. The Cdc7(⌬301–313) and Cdc7(⌬319 –326) mutants were localized in the nucleus, while Cdc7(⌬301–326) was mainly in the cytoplasm, suggesting amino acids 314 –318 is absolutely required for Cdc7 nuclear localization (Fig. 1, A and B).

Results

Conclusion