Cell cycle regulation of the replication licensing system: involvement of a Cdk-dependent inhibitor

H M Mahbubani, J P Chong, S Chevalier, P Thömmes, J J Blow

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Abstract

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.
Original languageEnglish
Pages (from-to)125-35
Number of pages11
JournalJournal of Cell Biology
Volume136
Issue number1
Publication statusPublished - 1997

Keywords

  • Adenine
  • Animals
  • Cell Cycle
  • Cell Cycle Proteins
  • Cyclin-Dependent Kinase Inhibitor p21
  • Cyclin-Dependent Kinases
  • Cyclins
  • Cycloheximide
  • DNA Replication
  • Enzyme Inhibitors
  • Protein Synthesis Inhibitors
  • Xenopus

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