DNA Induces Conformational Changes in a Recombinant Human Minichromosome Maintenance Complex * Received for publication, October 30, 2014, and in revised form, February 2, 2015Published, JBC Papers in Press, February 3, 2015, DOI 10.1074/jbc.M114.622738

Background: The human minichromosome maintenance (hMCM) complex is an important component of the DNA replication apparatus. Results: After being produced in Escherichia coli, hMCM has ATPase and DNA helicase activity and undergoes a conforma- tional change when bound to DNA. Conclusion: Recombinant hMCM is functional in vitro. Significance: hMCM provides an important tool for the biochemical reconstitution of the human replicative helicase. ATP-dependent DNA unwinding activity has been demon- stratedforrecombinantarchaealhomohexamericminichromo- some maintenance (MCM) complexes and their yeast hetero- hexameric counterparts, but in higher eukaryotes such as Drosophila, MCM-associated DNA helicase activity has been observed only in the context of a co-purified Cdc45-MCM- GINS complex. Here, we describe the production of the recom- binant human MCM (hMCM) complex in Escherichia coli. This protein displays ATP hydrolysis activity and is capable of unwinding duplex DNA. Using single-particle asymmetric EM reconstruction, we demonstrate that recombinant hMCM forms a hexamer that undergoes a conformational change when bound to DNA. Recombinant hMCM produced without post- translationalmodificationsisfunctionalinvitroandprovidesan importanttoolforbiochemicalreconstitutionofthehumanrep- licative helicase.