Isolation and characterization of various complexes of the minichromosome maintenance proteins of Schizosaccharomyces pombe

Minichromosome maintenance (Mcm) proteins 2-7 are highly conserved in eukar yotes and play an essential role in DNA replication. Here, we describe the reconstitution of the various complexes of the Mcm proteins of Schizosaccha romyces pombe using the baculovirus expression system. The simultaneous exp ression of all six of the Mcm proteins, as well as different combinations o f these proteins, yielded several stable complexes that included the hetero hexamer of Mcm2/3/4/5/6/7, the Mcm2/4/6/7 heterotetramer, the dimer of the Mcm4/6/7 heterotrimer, and the Mcm3/5 heterodimer, The purification and cha racterization of the biochemical properties of these complexes showed that only the dimeric complex of the Mcm4/6/7 heterotrimer possessed single stra nded DNA-dependent ATPase, ATP-dependent single stranded DNA binding, and 3 ' to 5' DNA helicase activities. Consistent with these results, the interac tion of either Mcm2 or Mcm3/5 with the Mcm4/6/7 complex resulted in the dis assembly of the dimeric complex of Mcm4/6/7 and the loss of DNA helicase ac tivity. These results suggest that the Mcm4/6/7 complex is a catalytic core of the Mcm complex and that Mcm2 and Mcm3/5 may be involved in the regulat ion of the activity of this complex.