Department of General Biology, School of Medicine, University of Patras, 26500 Rio, Patras, Greece.
J Cell Sci. 2011 Feb 1;124(Pt 3):422-34. doi: 10.1242/jcs.074229. Epub 2011 Jan 11.
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.
为了维持基因组完整性，细胞周期和 DNA 损伤反应必须联系在一起。Cdt1 是一种 G1 期特异性细胞周期因子，在 DNA 损伤后被 Cul4-Ddb1(Cdt2)泛素连接酶靶向蛋白酶解。使用激光纳米手术显微镜在活细胞核内产生空间限制的 DNA 损伤，我们发现 Cdt1 在 G1 期细胞中，在 DNA 损伤诱导后几秒钟内就被招募到损伤部位。PCNA、Cdt2、Cul4、DDB1 和 p21(Cip1)也迅速积累到损伤部位。Cdt1 的募集依赖于 PCNA，而 PCNA 和 Cdt2 的募集则不依赖于 Cdt1。荧光恢复后光漂白曲线的拟合分析反应-扩散模型表明，Cdt1 和 p21(Cip1)在损伤部位表现出高度动态的结合，而 PCNA 则表现出不移动。Cdt2 表现出快速交换和明显的不移动亚群。我们的数据表明，PCNA 为损伤部位的 Cdt1 动态相互作用提供了一个不移动的结合界面，导致 Cdt1 快速招募到受损 DNA，然后再进行 Cdt1 降解。
