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.