title: The physical interaction of Mcm10 with Cdc45 modulates their DNA-binding properties.
abstract: The eukaryotic DNA replication protein Mcm10 (mini-chromosome maintenance 10) associates with chromatin in early S-phase and is required for assembly and function of the replication fork protein machinery. Another essential component of the eukaryotic replication fork is Cdc45 (cell division cycle 45), which is required for both initiation and elongation of DNA replication. In the present study we characterize, for the first time, the physical and functional interactions of human Mcm10 and Cdc45. First we demonstrated that Mcm10 and Cdc45 interact in cell-free extracts. We then analysed the role of each of the Mcm10 domains: N-terminal, internal and C-terminal (NTD, ID and CTD respectively). We have detected a direct physical interaction between CTD and Cdc45 by both in vitro co-immunoprecipitation and surface plasmon resonance experiments. On the other hand, we have found that the interaction of the Mcm10 ID with Cdc45 takes place only in the presence of DNA. Furthermore, we found that the isolated ID and CTD domains are fully functional, retaining DNA-binding capability with a clear preference for bubble and fork structures, and that they both enhance Cdc45 DNA-binding affinity. The results of the present study demonstrate that human Mcm10 and Cdc45 directly interact and establish a mutual co-operation in DNA binding.
authors.affiliation: Istituto di Biochimica delle Proteine, Consiglio Nazionale delle Ricerche, Napoli, Italy.
authors.forename: Roberta
authors.initials: R
authors.lastname: Di Perna
authors.forename: Valentina
authors.initials: V
authors.lastname: Aria
authors.forename: Mariarosaria
authors.initials: M
authors.lastname: De Falco
authors.forename: Vincenzo
authors.initials: V
authors.lastname: Sannino
authors.forename: Andrei L
authors.initials: AL
authors.lastname: Okorokov
authors.forename: Francesca M
authors.initials: FM
authors.lastname: Pisani
authors.forename: Mariarita
authors.initials: M
authors.lastname: De Felice
mesh_terms: D001665:Binding Sites; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002474:Cell-Free System; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D057809:HEK293 Cells; D006801:Humans; D047468:Immunoprecipitation; D007700:Kinetics; D064110:Minichromosome Maintenance Proteins; D008958:Models, Molecular; D062105:Molecular Docking Simulation; D008970:Molecular Weight; D009690:Nucleic Acid Conformation; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D054730:Protein Interaction Domains and Motifs; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D020349:Surface Plasmon Resonance
chemical_list: C113571:CDC45 protein, human; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; C489613:MCM10 protein, human; D010446:Peptide Fragments; D011994:Recombinant Proteins; D004247:DNA; D064110:Minichromosome Maintenance Proteins