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 20 s while TopBP1 was retained at damage sites significantly longer suggesting different functions of the proteins during DSB recognition and repair.
Department of Cell and Molecular Biology, Leibniz Institute for Natural Product Research and Infection Biology (Hans Knoell Institute), Beutenbergstrasse 11a, D-07745 Jena, Germany.
Anja
A
Kuhnert
Uta
U
Schmidt
Shamci
S
Monajembashi
Claudia
C
Franke
Bernhard
B
Schlott
Frank
F
Grosse
Karl Otto
KO
Greulich
Hans-Peter
HP
Saluz
Frank
F
Hänel
D001483:Base Sequence; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D053903:DNA Breaks, Double-Stranded; D017931:DNA Primers / Q000235:genetics; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D034521:Nuclear Matrix-Associated Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D050810:Octamer Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000072670:PTB-Associated Splicing Factor; D054730:Protein Interaction Domains and Motifs; D040901:Proteomics; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020798:Two-Hybrid System Techniques
D002352:Carrier Proteins; D017931:DNA Primers; D004268:DNA-Binding Proteins; C083214:NONO protein, human; D034521:Nuclear Matrix-Associated Proteins; D009687:Nuclear Proteins; D050810:Octamer Transcription Factors; D000072670:PTB-Associated Splicing Factor; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; C110513:TOPBP1 protein, human