Replicative helicases are essential motor proteins that use nucleoside triphosphate-fueled conformational changes to unwind duplex DNA (reviewed in reference 25
). In eukaryotes, a ring-shaped heterohexameric ATPase called the m
aintenance (Mcm2-7) complex is believed to fulfill this role (reviewed in reference 12
). In combination with several other DNA replication factors (Cdc45 [31
] and the GINS complex [14
]), Mcm2-7 is physically present and functionally required for both the initiation and elongation phases of DNA replication (reviewed in reference 3
). These properties are reminiscent of the Escherichia coli
DNA replicative helicase DnaB (24
) and strongly implicate Mcm2-7, possibly in combination with Cdc45 or the GINS complex, as the eukaryotic replicative helicase (reviewed in reference 2
In contrast to homohexameric helicases, Mcm2-7 is formed from six different and individually essential subunits (numbered 2 to 7) that are all members of the AAA+
ATPase family (20
). This unique situation makes Mcm2-7 an attractive system for studying the coordination between ATP hydrolysis and motor function, since mutant complexes can be engineered with defined alterations at precise locations within the complex for study (e.g., see reference 29
). The differences between the six subunits are likely of functional significance, since each Mcm subunit forms a conserved and essential gene family dating back to the earliest evolutionary split between eukaryotes and the archaea (19
Although Mcm2-7 awaits structural analysis, precedence to other AAA+
members suggests that Mcm ATPase active sites are formed in trans
from a conserved Walker A and B motif from one subunit and a catalytically essential “arginine finger” from the other (11
). Five unique dimeric combinations likely corresponding to ATPase active sites have been identified (9
). For one dimer, composed of Mcm3 and Mcm7 (Mcm3/7), mutational analysis demonstrated that this dimer is a true active site with a typical AAA+
). However, it is unknown if the remaining dimers have a similar active-site arrangement.
Unlike a typical homohexameric helicase in which the six active sites participate equally (e.g., see references 10
), the ATPase active sites in Mcm2-7 may be functionally nonequivalent. Although historically Mcm2-7 lacks in vitro helicase activity, an Mcm subcomplex specifically containing only Mcm4, -6, and -7 (Mcm467 complex) has a weak helicase activity (18
). This observation differentiates between Mcm subunits involved in helicase activity (Mcm4, -6, and -7) and those that lack helicase activity (Mcm2, -3, and -5). In addition, the five putative Mcm active-site dimers have a wide range of ATPase activities (9
). However, both observations involve Mcm subcomplexes, and the relevance of these data to the intact Mcm2-7 hexamer is unknown.
To compare the activity of the isolated dimers to their corresponding activity within the Mcm2-7 hexamer, we generated and studied mutants within the six Walker B and arginine finger motifs. In addition to Mcm3/7, at least two additional active sites, Mcm7/4 and Mcm6/2, function in a combinatorial nature similar to that of other AAA+ proteins. Our data support a specific subunit architecture for Mcm2-7 that contains a physical discontinuity between Mcm2 and Mcm5. We also find a good correlation between the varied ATPase activity of isolated dimers and their contribution toward ATPase activity and viability within the Mcm2-7 hexamer. These data demonstrate that unlike the case with other hexameric helicases, active sites within Mcm2-7 contribute unequally toward activity.