Characterization of the interaction between the human DNA topoisomerase IIbeta-binding protein 1 (TopBP1) and the cell division cycle 45 (Cdc45) protein.
TopBP1 (topoisomerase IIbeta-binding protein 1) is a BRCT [BRCA1 (breast-cancer susceptibility gene 1) C-terminal]-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 DNA damage signalling. Experiments with fission yeast and Xenopus revealed that the TopBP1 homologues of these organisms are required for chromatin loading of the replication protein Cdc45 (cell division cycle 45). To improve our understanding of the physiological functions of human TopBP1, we investigated the interplay between human TopBP1 and Cdc45 proteins in synchronized HeLa-S3 cells. Using GST (glutathione transferase) pull-down and co-immunoprecipitation techniques, we showed a direct interaction between TopBP1 and Cdc45 in vitro and in vivo. The use of deletion mutants in GST pull-down assays identified the first and second as well as the sixth BRCT domains of TopBP1 to be responsible for the functional interaction with Cdc45. Moreover, the interaction between Cdc45 and the first and second BRCT domains of TopBP1 inhibited their transcriptional activation both in yeast and mammalian one-hybrid systems. Both proteins interacted exclusively at the G(1)/S boundary of cell cycle; only weak interaction could be found at the G(2)/M boundary. The overexpression of the sixth BRCT domain led to diminished loading of Cdc45 on to chromatin. These results suggest that human TopBP1 is involved in the formation of the initiation complex of replication in human cells and is required for the recruitment of Cdc45 to origins of DNA replication.
Schmidt
Uta
U
Department of Cell and Molecular Biology, Leibniz Institute for Natural Product Research and Infection Biology, Hans Knoell Institute, Beutenbergstrasse 11a, D-07745 Jena, Germany.
Wollmann
Yvonne
Y
Franke
Claudia
C
Grosse
Frank
F
Saluz
Hans-Peter
HP
Hänel
Frank
F
D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry*; D002453:Cell Cycle; D018797:Cell Cycle Proteins / Q000737:chemistry*; D002843:Chromatin / Q000378:metabolism; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000737:chemistry*; D005434:Flow Cytometry; D005982:Glutathione Transferase / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D009687:Nuclear Proteins / Q000737:chemistry*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013347:Subcellular Fractions; D020798:Two-Hybrid System Techniques; D014981:Xenopus
C113571:CDC45 protein, human; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D002843:Chromatin; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; C110513:TOPBP1 protein, human; D005982:Glutathione Transferase