Interaction and assembly of murine pre-replicative complex proteins in yeast and mouse cells.
Eukaryotic cells coordinate chromosome duplication by the assembly of protein complexes at origins of DNA replication by sequential binding of member proteins of the origin recognition complex (ORC), CDC6, and minichromosome maintenance (MCM) proteins. These pre-replicative complexes (pre-RCs) are activated by cyclin-dependent kinases and DBF4/CDC7 kinase. Here, we carried out a comprehensive yeast two-hybrid screen to establish sequential interactions between two individual proteins of the mouse pre-RC that are probably required for the initiation of DNA replication. The studies revealed multiple interactions among ORC subunits and MCM proteins as well as interactions between individual ORC and MCM proteins. In particular CDC6 was found to bind strongly to ORC1 and ORC2, and to MCM7 proteins. DBF4 interacts with the subunits of ORC as well as with MCM proteins. It was also demonstrated that CDC7 binds to different ORC and MCM proteins. CDC45 interacts with ORC1 and ORC6, and weakly with MCM3, -6, and -7. The three subunits of the single-stranded DNA binding protein RPA show interactions with various ORC subunits as well as with several MCM proteins. The data obtained by yeast two-hybrid analysis were paradigmatically confirmed in synchronized murine FM3A cells by immunoprecipitation of the interacting partners. Some of the interactions were found to be cell-cycle-dependent; however, most of them were cell-cycle-independent. Altogether, 90 protein-protein interactions were detected in this study, 52 of them were found for the first time in any eukaryotic pre-RC. These data may help to understand the complex interplay of the components of the mouse pre-RC and should allow us to refine its structural architecture as well as its assembly in real time.
Kneissl
Margot
M
Institute of Biochemistry, University of Würzburg, Biozentrum Am Hubland, D-97074 Würzburg, Germany.
Pütter
Vera
V
Szalay
Aladar A
AA
Grummt
Friedrich
F
D000818:Animals; D002453:Cell Cycle; D018797:Cell Cycle Proteins / Q000378:metabolism; D002460:Cell Line; D004261:DNA Replication / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000378:metabolism; D006801:Humans; D046911:Macromolecular Substances; D051379:Mice; D064168:Minichromosome Maintenance Complex Component 7; D009687:Nuclear Proteins / Q000378:metabolism; D051738:Origin Recognition Complex; D011233:Precipitin Tests; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D021122:Protein Subunits / Q000737:chemistry* / Q000378:metabolism*; D018741:Replication Origin / Q000502:physiology*; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D020798:Two-Hybrid System Techniques
C066503:CDC6 protein, S cerevisiae; D018797:Cell Cycle Proteins; C485678:DBF4 protein, human; C121983:DBF4 protein, mouse; D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D009687:Nuclear Proteins; C497214:ORC1 protein, S cerevisiae; C497223:ORC1 protein, human; C497215:ORC2 protein, S cerevisiae; C497221:ORC6 protein, S cerevisiae; C497228:Orc1 protein, mouse; D051738:Origin Recognition Complex; D021122:Protein Subunits; D029701:Saccharomyces cerevisiae Proteins; C491088:MCM7 protein, S cerevisiae; C101624:MCM7 protein, human; C491086:Mcm7 protein, mouse; D064168:Minichromosome Maintenance Complex Component 7