title: Mutations in subdomain B of the minichromosome maintenance (MCM) helicase affect DNA binding and modulate conformational transitions.
abstract: Minichromosome maintenance (MCM) proteins are believed to provide the replicative helicase activity in eukaryotes and archaea. The single MCM orthologue from Methanothermobacter thermautotrophicus (MthMCM) has been extensively characterized as a model of the eukaryotic heterohexameric MCM complex. MthMCM forms high molecular weight complexes in solution consistent with a dodecamer. Visualization of this complex by electron microscopy suggests that single and double heptameric or hexameric rings can form. We have mutated two arginine residues (Arg-137, Arg-160) in the N-terminal subdomain B of MthMCM based on their apparent potential to form inter-ring hydrogen bonds. Both the single R137A and the double R137A,R160A mutants were characterized by a combination of biophysical, biochemical, and electron microscopy techniques. Biophysical analysis coupled with electron microscopy studies shows that the R137A mutant forms a double heptameric ring, whereas the R137A,R160A protein assembles as a single heptamer. They both show a defect in DNA binding and a concomitant conformational change in subdomain A, with the double mutant displaying significant defects in helicase activity as well. We propose a model in which MCM loading and the subsequent activation of the helicase activity involve a conformational transition that is connected to a DNA binding event.
authors.affiliation: Department of Biology and Technology Facility, University of York, York YO10 5YW, United Kingdom.
authors.forename: Elizabeth R
authors.initials: ER
authors.lastname: Jenkinson
authors.forename: Alessandro
authors.initials: A
authors.lastname: Costa
authors.forename: Andrew P
authors.initials: AP
authors.lastname: Leech
authors.forename: Ardan
authors.initials: A
authors.lastname: Patwardhan
authors.forename: Silvia
authors.initials: S
authors.lastname: Onesti
authors.forename: James P J
authors.initials: JP
authors.lastname: Chong
mesh_terms: D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004265:DNA Helicases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D019641:DNA, Archaeal / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins; D017013:Methanobacteriaceae / Q000201:enzymology* / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics*; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D014461:Ultracentrifugation
chemical_list: D019843:Archaeal Proteins; D019641:DNA, Archaeal; D004268:DNA-Binding Proteins; D004265:DNA Helicases; C403162:MCM protein, Methanobacterium thermoautotrophicum