• Authors: Mortusewicz, O., Leonhardt, H.
  • Year: 2007
  • Journal: BMC Mol Biol 8 81
  • Applications: in vitro / DNA / jetPEI
  • Cell type: HeLa
    Description: Human cervix epitheloid carcinoma cells


BACKGROUND: Genome integrity is constantly challenged and requires the coordinated recruitment of multiple enzyme activities to ensure efficient repair of DNA lesions. We investigated the dynamics of XRCC1 and PCNA that act as molecular loading platforms and play a central role in this coordination. RESULTS: Local DNA damage was introduced by laser microirradation and the recruitment of fluorescent XRCC1 and PCNA fusion proteins was monitored by live cell microscopy. We found an immediate and fast recruitment of XRCC1 preceding the slow and continuous recruitment of PCNA. Fluorescence bleaching experiments (FRAP and FLIP) revealed a stable association of PCNA with DNA repair sites, contrasting the high turnover of XRCC1. When cells were repeatedly challenged with multiple DNA lesions we observed a gradual depletion of the nuclear pool of PCNA, while XRCC1 dynamically redistributed even to lesions inflicted last. CONCLUSION: These results show that PCNA and XRCC1 have distinct kinetic properties with functional consequences for their capacity to respond to successive DNA damage events.