The physical interaction of Mcm10 with Cdc45 modulates their DNA binding properties

The eukaryotic DNA replication protein Mcm10 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, which is required for both initiation and elongation of DNA replication. Here, we characterize for the first time the physical and functional interactions of human Mcm10 and Cdc45. We first demonstrated that Mcm10 and Cdc45 interact cell free extracts. We then analyzed 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 interaction of 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. Our results demonstrate that human Mcm10 and Cdc45 directly interact and establish a mutual cooperation in DNA binding.

Source: BJ Gene - June 10, 2013 Category: Biochemistry Authors: R Di Perna, V Aria, M De Falco, V Sannino, A L Okorokov, F M Pisani, M De Felice Tags: BJ Gene Source Type: research