MCM-BP is known to make important contributions to DNA replication presumably through its interaction with MCM complexes, although the nature of these interactions have not been well defined. Here we have shown that MCM-BP has some propensity to interact with any of the MCM 2 to 7 proteins, although it appears to interact most stably with MCM4 and MCM7. These interactions can lead to the dissociation of MCM2-7 hexamers in vitro and can affect the solubility or behaviour of some individual MCM proteins. We also identified an interaction with the Dbf4 regulatory component of the DDK kinase, known to phosphorylate MCM proteins and to play multiple roles in DNA replication.
Assessment of the physical interactions of MCM-BP with MCM proteins expressed in insect cells indicated that, while MCM-BP can interact with any individual MCM protein, it interacts most strongly with MCM4 and MCM7. A preferential interaction with MCM7, but not MCM4, was previously reported for Xenopus
MCM-BP. Sucrose gradient analysis of Xenopus
egg extracts identified a complex containing MCM-BP and MCM7, but not other MCMs, suggesting preferential association of MCM-BP with MCM7 
. Furthermore, the interaction between MCM-BP and MCM7 was shown in GST pulldown assays to involve the MCM box of MCM7 
. Since the MCM box is conserved in all MCM proteins, this finding fits with our observations that MCM-BP has some capacity to interact with all MCM proteins. The interaction between Xenopus
MCM-BP and MCM7 was also found to be stable up to 0.8 M NaCl, consistent with our findings that MCM-BP interactions with MCM proteins are very salt stable. This is typical of interactions between MCM proteins which remain intact in up to 1 M NaCl 
By assaying pairwise MCM protein interactions and determining those that result in ATPase activity, the order of the MCM proteins in the hexameric ring has been determined 
. Further work has suggested that a ‘gate’ exists between MCM2 and MCM5 whose state dictates the open and closed conformations of the MCM complex 
. A ‘closed’ MCM 2/5 gate has been shown to promote the helicase activity of the MCM complex by activating the MCM4/7 motor, located 180 degrees across from the MCM2/5 gate. Our finding that MCM-BP binds most prominently to MCM4 and MCM7 suggests that MCM-BP may contact the hexameric ring through these proteins across from the gate, and further regulate helicase activity. Considerable evidence indicates that MCM4 and MCM7 are key proteins in the MCM complex and important targets for MCM functional regulation. Phosphorylation of MCM4 serves as a means of regulating the MCM complex, as MCM4 phosphorylation by cyclin A/Cdk2 inactivates the helicase activity 
. MCM7 is also of particular importance for the activity of the MCM2-7 hexamer as it contributes to two ATPase active sites (with MCM4 and MCM3) 
. In addition, protein interactions with MCM7 are known to regulate the activity of the MCM complex. For example, an interaction between cyclin A and MCM7 promotes S-phase entry 
, while DNA replication is inhibited by binding of the retinoblastoma protein to MCM7 
. MCM7 also appears to have functions independent of the MCM complex, as MCM7 (but not other MCM proteins) is important for Chk1 signalling through its interactions with Rad17 and ATR-interacting protein (ATRIP) 
. In addition, a role for MCM7 in hypoxia was recently identified in which MCM7 binds and induces the degradation of hypoxia-inducible factor 1 (HIF-1) 
. Therefore, in addition to dissociating the MCM complex, MCM-BP interactions with MCM7 and MCM4 may regulate the functions of the MCM complex as well as other roles of these proteins.
Our finding that MCM-BP can affect the solubility of some individual MCM proteins raises the possibility that MCM-BP could serve a chaperone-like function affecting the pool of MCM proteins that are not assembled on chromatin. This property of MCM-BP might also be important for promoting the dissociation of MCM complexes. Previous studies on the Xenopus
and S. pombe
versions of MCM-BP supported a role for MCM-BP in dissociating MCM hexamers 
. We have now shown that this property is also intrinsic to human MCM-BP and that MCM complexes from either G1 (the dominant phase in log-phase cells) or various stages of S-phase can be disrupted by MCM-BP.
The co-purification of MCMs3-7 with MCM-BP from a variety of organisms suggests that MCM-BP can form a hexameric complex with these MCM proteins 
. In addition, MCM-BP can form a complex with the MCM4,6,7 core helicase when co-expressed with them in insect cells 
. However, examination of the state of MCM-BP complexes by glycerol or sucrose gradient sedimentation of cell extracts has given variable results in different organisms. Analysis of Xenopus
interphase egg extracts detected a complex of MCM-BP and MCM7 but did not detect MCM-BP in large MCM complexes 
. Li et al 
found that pombe
Mcb1 co-migrated with MCM4 and MCM6 but the migration of other MCM proteins was not examined. Our analysis of human cell extracts indicated that most of the MCM-BP did not co-migrate with the MCM hexamers, nor did we detect obvious complexes between MCM-BP and single MCM subunits. However, a small proportion of the MCM-BP appeared as a distinct high-molecular weight peak in mid to late S phase, that also contained the MCM proteins. This observation may be relevant for MCM complex unloading since previous data suggests that MCM-BP promotes the dissociation of MCM complexes from the chromatin in mid to late S 
. Our results suggest that, in human cells, higher order complexes between MCM-BP and MCM proteins are not constitutive but may form transiently during S phase.
Interactions between MCM proteins and DDK are known to be important for origin activation. Since MCM-BP forms complexes with MCM proteins and can be detected on cellular origins at G1/S (when DDK is active) 
, we wanted to examine whether MCM-BP interacted with the Dbf4 regulatory component of this kinase. We found that MCM-BP interacted with Dbf4 in yeast 2-hybrid assays and upon co-expression in insect cells, and that the two endogenous proteins co-immunoprecipitated from human cells. While the interaction of DDK with the MCM complex is known to result in the phosphorylation of MCM2, MCM4 and MCM6, an important event in origin activation 
, we found that MCM-BP was not phosphorylated by DDK in vitro
whereas MCM2, 4, 6 and 7 were phosphorylated under the same conditions. While MCM7 phosphorylation by DDK has not been widely studied, budding yeast MCM7 has also been reported to be a substrate for DDK 
. Interestingly, we found that MCM-BP inhibited DDK phosphorylation of the MCM4,6,7 complex in a dose-dependent manner, suggesting that the interaction of MCM-BP with DDK and/or the MCM4,6,7 complex interfered with phosphorylation. Indeed we have previously shown that MCM-BP forms a complex with MCM4,6,7 that is stable to glycerol gradient sedimentation 
. The finding that DDK phosphorylation of MCM2 was less affected by MCM-BP suggests that the strong interaction of MCM-BP with the MCM4,6,7 complex is at least partly responsible for this inhibition, as opposed to having a direct effect on cdc7 activity. We also confirmed these findings in MCM2-7 hexamers, where MCM-BP had little effect on MCM2 but inhibited DDK phosphorylation of one or more of the other MCM proteins. The ability of MCM-BP to affect DDK phosphorylation of MCM 4 and 6 may be relevant for origin activation where these phosphorylation events have been shown to be important. MCM-BP was found to be preferentially associated with the lamin B2 origin at G1/S where it could conceivably influence DDK phosphorylation at this stage of the cell cycle 
. In addition, DDK has been found to be important for S-phase progression through MCM4 phosphorylation, Chk1 checkpoint signalling and replication fork restart after a prolonged S-phase checkpoint, raising the possibility that MCM-BP might also impact these processes through regulation of DDK phosphorylation 
. Future studies on the role of the MCM-BP/Dbf4 interaction will be important for elucidating the mechanism of action of MCM-BP and its functions in DNA replication.