Proteomic identification of PSF and p54(nrb) as topBP1-interacting proteins

TopBP1 is a BRCT domain‐rich protein that is structurally and functionally conserved throughout eukaryotic organisms. It is required for the initiation of DNA replication and for DNA repair and damage signalling. To further dissect its biological functions, we explored TopBP1‐interacting proteins by co‐immunoprecipitation assays and LC‐ESI‐MS‐analyses. As TopBP1 binding partners we identified p54(nrb) and PSF, and confirmed the physical interactions by GST pull‐down assays, co‐immunoprecipitations and by yeast two‐hybrid experiments. Recent evidence shows an involvement of p54(nrb) and PSF in DNA double‐strand break repair (DSB) and radioresistance. To get a first picture of the physiological significance of the interaction of TopBP1 with p54(nrb) and PSF we investigated in real time the spatiotemporal behaviour of the three proteins after laser microirradiation of living cells. Localisation of TopBP1 at damage sites was noticed as early as 5 s following damage induction, whereas p54(nrb) and PSF localised there after 20 s. Both p54(nrb) and PSF disappeared after 200 s while TopBP1 was retained at damage sites significantly longer suggesting different functions of the proteins during DSB recognition and repair. J. Cell. Biochem. 113: 1744–1753, 2012. © 2012 Wiley Periodicals, Inc.