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© The Rockefeller University Press, 0021-9525/1997//125 $5.00
The Journal of Cell Biology, Volume 136, Number 1, , 1997 125-135

Article

Cell Cycle Regulation of the Replication Licensing System: Involvement of a Cdk-dependent Inhibitor



Hiro M. Mahbubani, James P.J. Chong, Stephane Chevalier, Pia Thömmes, and J. Julian Blow

Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms, Potters Bar, Herts EN6 3LD, United Kingdom

The replication licensing factor (RLF) is an essential initiation factor that is involved in preventing re-replication of chromosomal DNA in a single cell cycle. In Xenopus egg extracts, it can be separated into two components: RLF-M, a complex of MCM/P1 polypeptides, and RLF-B, which is currently unpurified. In this paper we investigate variations in RLF activity throughout the cell cycle. Total RLF activity is low in metaphase, due to a lack of RLF-B activity and the presence of an RLF inhibitor. RLF-B is rapidly activated on exit from metaphase, and then declines during interphase. The RLF inhibitor present in metaphase extracts is dependent on the activity of cyclin-dependent kinases (Cdks). Affinity depletion of Cdks from metaphase extracts removed the RLF inhibitor, while Cdc2/cyclin B directly inhibited RLF activity. In metaphase extracts treated with the protein kinase inhibitor 6-dimethylaminopurine (6-DMAP), both cyclin B and the RLF inhibitor were stabilized although the extracts morphologically entered interphase. These results are consistent with studies in other organisms that invoke a key role for Cdks in preventing re-replication of DNA in a single cell cycle.

Abbreviations used in this paper: BrdUTP, bromodeoxyuridine triphosphate; Cdk, cyclin-dependent kinase; 6-DMAP, 6-dimethyl aminopurine; GST, glutathione-S-transferase; MPF, maturation promoting factor; ORC, origin recognition complex; PEG, polyethylene glycol; RLF, replication licensing factor; SPF, S-phase promoting factor.

J.J. Blow is a Lister Institute Research fellow. We thank Noel Lowndes and John Diffley for comments on the manuscript.

Received for publication 16 August 1996 and in revised form 23 October 1996.

Address all correspondence to J. Julian Blow, Imperial Cancer Research Fund, Clare Hall Laboratories, Blanche Lane, South Mimms, Potters Bar, Herts EN6 3LD, United Kingdom. Tel.: (44) 171-269-3923. Fax: (44) 171269-3801. e-mail: blow{at}icrf.icnet.uk


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