Article
- The EMBO Journal (2007) 26, 1303 - 1314
- doi:10.1038/sj.emboj.7601597
Published online: 22 February 2007
Subject Category:
Cdt1 associates dynamically with chromatin throughout G1 and recruits Geminin onto chromatin
Georgia Xouri1, Anthony Squire2, Maria Dimaki1, Bart Geverts3, Peter J Verveer2, Stavros Taraviras4, Hideo Nishitani5, Adriaan B Houtsmuller3, Philippe I H Bastiaens2 and Zoi Lygerou1
- Laboratory of General Biology, School of Medicine, University of Patras, Patras, Greece
- Cell Biology and Biophysics Unit, EMBL, Heidelberg, Germany
- Josephine Nefkens Institute, Erasmus MC, University Medical Centre Rotterdam, Rotterdam, The Netherlands
- Laboratory of Pharmacology, School of Medicine, University of Patras, Patras, Greece
- Department of Molecular Biology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan
Correspondence to:
Zoi Lygerou, Laboratory of General Biology, School of Medicine, University of Patras, University Campus, Rio, Patras 26500, Greece. Tel.: + 30 2610 997621; Fax: + 30 2610 991769; E-mail: lygerou@med.upatras.gr
Philippe I H Bastiaens, Cell Biology and Biophysics Unit, EMBL, Heidelberg 69117, Germany. Tel.: + 49 6221 387 407; Fax: + 49 6221 387 512; E-mail: bastiaen@embl.de
Received 6 June 2006; Accepted 9 January 2007
Abstract
To maintain genome integrity, eukaryotic cells initiate DNA replication once per cell cycle after assembling prereplicative complexes (preRCs) on chromatin at the end of mitosis and during G1. In S phase, preRCs are disassembled, precluding initiation of another round of replication. Cdt1 is a key member of the preRC and its correct regulation via proteolysis and by its inhibitor Geminin is essential to prevent premature re-replication. Using quantitative fluorescence microscopy, we study the interactions of Cdt1 with chromatin and Geminin in living cells. We find that Cdt1 exhibits dynamic interactions with chromatin throughout G1 phase and that the protein domains responsible for chromatin and Geminin interactions are separable. Contrary to existing in vitro data, we show that Cdt1 simultaneously binds Geminin and chromatin in vivo, thereby recruiting Geminin onto chromatin. We propose that dynamic Cdt1–chromatin associations and the recruitment of Geminin to chromatin provide spatio-temporal control of the licensing process.
Keywords:
- Cdt1,
- fluorescence lifetime imaging microscopy (FLIM),
- fluorescence recovery after photobleaching (FRAP),
- Geminin,
- licensing
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