Cdc6 Chromatin Affinity Is Unaffected by Serine-54 Phosphorylation, S-Phase Progression, and Overexpression of Cyclin A

Abstract

Ectopically expressed Cdc6 is translocated from the nucleus during S phase in a cyclin A-Cdk2-dependent process, suggesting that reinitiation of DNA replication is prevented by removal of phosphorylated Cdc6 from chromatin after origin firing. However, whether endogenous Cdc6 translocates during S phase remains controversial. To resolve the questions regarding regulation of endogenous Cdc6, we cloned the cDNA encoding the Chinese hamster Cdc6 homolog and specifically focused on analyzing the localizations and chromatin affinities of endogenous and exogenous proteins during S phase and following overexpression of cyclin A. In agreement with other reports, ectopically expressed Cdc6 translocates from the nucleus during S phase and in response to overexpressed cyclin A. In contrast, using a combination of biochemical and immunohistochemical assays, we show convincingly that endogenous Cdc6 remains nuclear and chromatin bound throughout the entire S period, while Mcm5 loses chromatin affinity during S phase. Overexpression of cyclin A is unable to alter the nuclear localization of Cdc6. Furthermore, using a phosphospecific antibody we show that phosphoserine-54 Cdc6 maintains a high affinity for chromatin during the S period. Considering recent in vitro studies, these data are consistent with a proposed model in which Cdc6 is serine-54 phosphorylated during S phase and functions as a chromatin-bound signal that prevents reformation of prereplication complexes.

We thank Nicholas Heintz (University of Vermont) for the anti-Cdc6 monoclonal antibodies, Rolf Knippers (University of Konstanz) for anti-Mcm5 antibody, Joseph Nevins (Duke University) for the cyclin A plasmid, and Rong Li (University of Virginia) for plasmid pRcLac. We also thank Carlton White for technical assistance and the other members of the Hamlin laboratory for valuable discussions.

This work was supported by National Institutes of Health grant RO1 GM 26108 (to J.L.H.) and National Institutes of Health postdoctoral fellowship 5F32 GM19304-02 (to M.G.A.).