Dynamic recruitment of licensing factor Cdt1 to sites of DNA damage

Roukos, V. and Kinkhabwala, A. and Colombelli, J. and Kotsantis, P. and Taraviras, S. and Nishitani, H. and Stelzer, E. and Bastiaens, P. and Lygerou, Z. (2011) Dynamic recruitment of licensing factor Cdt1 to sites of DNA damage. Journal of Cell Science, 124 (3). pp. 422-434. ISSN 0021-9533

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-Ddb1Cdt2 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 p21Cip1 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 p21Cip1 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.