Cdt1 associates dynamically with chromatin throughout G1 and recruits Geminin onto chromatin.
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.
Xouri
Georgia
G
Laboratory of General Biology, School of Medicine, University of Patras, Patras, Greece.
Squire
Anthony
A
Dimaki
Maria
M
Geverts
Bart
B
Verveer
Peter J
PJ
Taraviras
Stavros
S
Nishitani
Hideo
H
Houtsmuller
Adriaan B
AB
Bastiaens
Philippe I H
PI
Lygerou
Zoi
Z
D002453:Cell Cycle; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D002843:Chromatin / Q000378:metabolism*; D036681:Fluorescence Recovery After Photobleaching / Q000379:methods; D016193:G1 Phase; D064248:Geminin; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008856:Microscopy, Fluorescence / Q000379:methods; D008954:Models, Biological; D009010:Monte Carlo Method; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
C467199:CDT1 protein, human; D018797:Cell Cycle Proteins; D002843:Chromatin; C577995:GMNN protein, human; D064248:Geminin; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins