Assembly of a homohexameric minichromosome maintenance complex is dependent on ATP and DNA.

The minichromosome maintenance (MCM) complex is the replicative helicase in eukaryotes and archaea, unwinding genomic DNA upstream of DNA polymerase. The eukaryotic MCM complex forms from six different subunits (Mcm2-7), whereas in archaea, the complex is homohexameric. Both types of MCM can assemble into functional helicases in vitro in the absence of cofactors. However, despite being simpler in composition, we know little about how a homohexameric archaeal MCM assembles, largely because the field has lacked a convenient system to interrogate. Historically, characterization of archaeal MCMs has focused on proteins from thermophilic organisms, which typically form robust oligomers in solution. We have identified an uncharacterized MCM from the mesophilic archaeon Mancarchaeum acidophilum (MacMCM) that shows strong DNA unwinding activity at room temperature. Unexpectedly, apo-MacMCM is monomeric in solution, providing a first opportunity to investigate the mechanisms of assembly of an active homohexameric MCM complex in vitro. We show that MacMCM requires both ATP and DNA to form an active homohexamer, and that the C-terminal winged-helix domain impedes oligomerization. We report the 3D structure of MacMCM, which reveals similar numbers of interactions at subunit-subunit interfaces as eukaryotic MCMs but fewer than MCMs from thermophilic archaea. Finally, we show that installing subunit-subunit salt bridges from Sulfolobus solfataricus MCM into MacMCM promotes oligomerization. Heterohexameric eukaryotic MCMs evolved from a homomeric ancestor. Our results identify structural and ligand-driven mechanisms of assembly that are conserved between homomeric and heteromeric MCMs.