title: Dynamic recruitment of licensing factor Cdt1 to sites of DNA damage.
abstract: For genomic integrity to be maintained, the cell cycle and DNA damage responses must be linked. Cdt1, a G1-specific cell-cycle factor, is targeted for proteolysis by the Cul4-Ddb1(Cdt2) ubiquitin ligase following DNA damage. Using a laser nanosurgery microscope to generate spatially restricted DNA damage within the living cell nucleus, we show that Cdt1 is recruited onto damaged sites in G1 phase cells, within seconds of DNA damage induction. PCNA, Cdt2, Cul4, DDB1 and p21(Cip1) also accumulate rapidly to damaged sites. Cdt1 recruitment is PCNA-dependent, whereas PCNA and Cdt2 recruitment are independent of Cdt1. Fitting of fluorescence recovery after photobleaching profiles to an analytic reaction-diffusion model shows that Cdt1 and p21(Cip1) exhibit highly dynamic binding at the site of damage, whereas PCNA appears immobile. Cdt2 exhibits both a rapidly exchanging and an apparently immobile subpopulation. Our data suggest that PCNA provides an immobile binding interface for dynamic Cdt1 interactions at the site of damage, which leads to rapid Cdt1 recruitment to damaged DNA, preceding Cdt1 degradation.
authors.affiliation: Department of General Biology, School of Medicine, University of Patras, 26500 Rio, Patras, Greece.
authors.forename: Vassilis
authors.initials: V
authors.lastname: Roukos
authors.forename: Ali
authors.initials: A
authors.lastname: Kinkhabwala
authors.forename: Julien
authors.initials: J
authors.lastname: Colombelli
authors.forename: Panagiotis
authors.initials: P
authors.lastname: Kotsantis
authors.forename: Stavros
authors.initials: S
authors.lastname: Taraviras
authors.forename: Hideo
authors.initials: H
authors.lastname: Nishitani
authors.forename: Ernst
authors.initials: E
authors.lastname: Stelzer
authors.forename: Philippe
authors.initials: P
authors.lastname: Bastiaens
authors.forename: Zoi
authors.initials: Z
authors.lastname: Lygerou
mesh_terms: D018797:Cell Cycle Proteins / Q000502:physiology*; D045744:Cell Line, Tumor; D044842:Cullin Proteins / Q000502:physiology; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000502:physiology; D004249:DNA Damage / Q000502:physiology* / Q000528:radiation effects; D004260:DNA Repair / Q000502:physiology; D004268:DNA-Binding Proteins / Q000502:physiology; D005347:Fibroblasts / Q000502:physiology; D016193:G1 Phase / Q000502:physiology; D006801:Humans; D009687:Nuclear Proteins / Q000502:physiology; D018809:Proliferating Cell Nuclear Antigen / Q000502:physiology*; D044767:Ubiquitin-Protein Ligases / Q000502:physiology; D014466:Ultraviolet Rays
chemical_list: C467199:CDT1 protein, human; D018797:Cell Cycle Proteins; D044842:Cullin Proteins; D050759:Cyclin-Dependent Kinase Inhibitor p21; C091435:DDB1 protein, human; D004268:DNA-Binding Proteins; C429530:DTL protein, human; D009687:Nuclear Proteins; D018809:Proliferating Cell Nuclear Antigen; D044767:Ubiquitin-Protein Ligases