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.

An intact Mcm10 coiled-coil interaction surface is important for origin melting, helicase assembly and the recruitment of Pol-α to Mcm2–7

“…Mutations at specific residues of NTD CC region disrupted the self-association of Mcm10 in vitro and in vivo (36). Oligomerization has also been observed in human Mcm10 (41), consistent with a hexameric ring structure shown by electron microscopy (42). The Mcm10 NTD has also been implicated in the interaction of Mcm10 with the Mec3 subunit of the 9-1-1 clamp, in a region distinct from Mcm10's self-interaction domain (amino acids 100–150).…”

MCM10: One tool for all—Integrity, maintenance and damage control

“…Removal of the corresponding motif in ScMcm10 leads to severe hydroxyurea (HU) sensitivity when checkpoint function is compromised [8](Alver and Bielinsky, unpublished). The NTD has also been implicated in oligomerization of human (Hs) Mcm10 [16]. Gel-filtration of this domain identified a high-molecular weight complex that was interpreted to be either a trimer or hexamer [16].…”

“…The NTD has also been implicated in oligomerization of human (Hs) Mcm10 [16]. Gel-filtration of this domain identified a high-molecular weight complex that was interpreted to be either a trimer or hexamer [16]. HsMcm10 has been reported to form a hexameric ring based on electron microscopy (EM) reconstruction of full-length protein in which the ring-shaped archaeal Mcm helicase was utilized for molecular modeling [17].…”

“…These results strongly imply that the Zn-F1 domain binds DNA in vivo . Lastly, there is evidence that the ID of Mcm10 and Cdc45 bind DNA cooperatively [16]. …”

“…The Masukata laboratory has provided some evidence that Mcm10's ability to bind ssDNA is important in this context [27]. It is likely however, that additional properties of Mcm10 make it the ideal tool for this task, as it has been shown to interact with Cdc45 [16, 30, 31], distinct subunits of Mcm2-7 and GINS [2, 20, 30, 32–34], ORC [30, 33–35], the Sld2 ortholog Recql4 [36] and the ssDNA binding complex replication protein A (RPA) [5]. Although RPA has a 40-fold higher affinity for ssDNA than Mcm10 [13], the latter also binds dsDNA [10, 12, 13], uniquely enabling it to bind parental and unwound DNA.…”