title: The Mcm2-7 complex has in vitro helicase activity.
abstract: Helicases unwind duplex DNA ahead of the polymerases at the replication fork. However, the identity of the eukaryotic replicative helicase has been controversial; in vivo studies implicate the ring-shaped heterohexameric Mcm2-7 complex, although only a specific subset of Mcm subunits (Mcm467) unwind DNA in vitro. To address this discrepancy, we have compared both Mcm assemblies and find that they differ in their linear single-stranded DNA association rate and their ability to bind circular single-stranded DNA. These differences depend upon the Mcm2/5 interface, which we hypothesize serves as an ATP-dependent "gate" within Mcm2-7. Importantly, we find that reaction conditions that putatively close the Mcm2-7 "gate" reconstitute Mcm2-7 helicase activity. Unlike Mcm467, Mcm2-7 helicase activity is strongly anion dependent. Our results show that purified Mcm2-7 acts as a helicase, provides functional evidence of a Mcm2/5 gate, and lays the foundation for future mechanistic studies of this critical factor.
authors.affiliation: Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260, USA.
authors.forename: Matthew L
authors.initials: ML
authors.lastname: Bochman
authors.forename: Anthony
authors.initials: A
authors.lastname: Schwacha
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism; D001665:Binding Sites; D018797:Cell Cycle Proteins / Q000378:metabolism*; D004265:DNA Helicases / Q000378:metabolism*; D004270:DNA, Circular / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D018698:Glutamic Acid / Q000494:pharmacology; D007700:Kinetics; D046912:Multiprotein Complexes / Q000378:metabolism*; D009154:Mutation / Q000235:genetics; D011485:Protein Binding / Q000187:drug effects; D021122:Protein Subunits / Q000378:metabolism
chemical_list: D018797:Cell Cycle Proteins; D004270:DNA, Circular; D004277:DNA, Single-Stranded; D046912:Multiprotein Complexes; D021122:Protein Subunits; D018698:Glutamic Acid; D000251:Adenosine Triphosphatases; D004265:DNA Helicases