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

• Published in: The Journal of cell biology Vol. 136; no. 1; pp. 125 - 135
• Main Authors: Mahbubani, Hiro M., James P. J. Chong, Chevalier, Stephane, Thömmes, Pia, Blow, J. Julian
• Format: Journal Article
• Language: English
• Published: United States Rockefeller University Press 13.01.1997; The Rockefeller University Press
• Subjects: Adenine - analogs & derivatives; Adenine - pharmacology; Animals; Cell cycle; Cell Cycle - physiology; Cell Cycle Proteins - metabolism; Cell Cycle Proteins - pharmacology; Cell Cycle Proteins - physiology; Cell division; Cell free system; Cellular biology; Chromatin; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinases - antagonists & inhibitors; Cyclin-Dependent Kinases - physiology; Cyclins; Cyclins - pharmacology; Cycloheximide - pharmacology; DNA; DNA replication; DNA Replication - drug effects; DNA Replication - physiology; Enzyme Inhibitors - pharmacology; Gels; Interphase; Metaphase; Ova; Protein Synthesis Inhibitors - pharmacology; Xenopus
• ISSN: 0021-9525; 1540-8140
• DOI: 10.1083/jcb.136.1.125

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.