Mutations in Subdomain B of the Minichromosome Maintenance (MCM) Helicase Affect DNA Binding and Modulate Conformational Transitions

TY - JOUR

T1 - Mutations in Subdomain B of the Minichromosome Maintenance (MCM) Helicase Affect DNA Binding and Modulate Conformational Transitions

AU - Jenkinson, Elizabeth R.

AU - Costa, Alessandro

AU - Leech, Andrew P.

AU - Patwardhan, Ardan

AU - Onesti, Silvia

AU - Chong, James P. J.

PY - 2009/2/27

Y1 - 2009/2/27

N2 - 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.

AB - 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.

KW - METHANOTHERMOBACTER-THERMAUTOTROPHICUS MCM

KW - METHANOBACTERIUM-THERMOAUTOTROPHICUM MCM

KW - SACCHAROMYCES-CEREVISIAE

KW - PROTEIN COMPLEX

KW - REPLICATION

KW - POLYMORPHISM

KW - MICROSCOPY

KW - INITIATION

KW - SYMMETRY

KW - MOTIFS

UR - http://www.scopus.com/inward/record.url?scp=64149103213&partnerID=8YFLogxK

U2 - 10.1074/jbc.M806973200

DO - 10.1074/jbc.M806973200

M3 - Article

SN - 0021-9258

VL - 284

SP - 5654

EP - 5661

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

IS - 9

ER -