Mutations in subdomain B of the minichromosome maintenance (MCM) helicase affect DNA binding and modulate conformational transitions.
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.
Jenkinson
Elizabeth R
ER
Department of Biology and Technology Facility, University of York, York YO10 5YW, United Kingdom.
Costa
Alessandro
A
Leech
Andrew P
AP
Patwardhan
Ardan
A
Onesti
Silvia
S
Chong
James P J
JP
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
D019843:Archaeal Proteins; D019641:DNA, Archaeal; D004268:DNA-Binding Proteins; D004265:DNA Helicases; C403162:MCM protein, Methanobacterium thermoautotrophicum