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

Elizabeth R Jenkinson,Alessandro Costa,Andrew P Leech,Ardan Patwardhan,Silvia Onesti,James P.J Chong

doi:10.1074/jbc.m806973200

Elizabeth R Jenkinson, Alessandro Costa + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m806973200

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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.

Highlights

The process of DNA replication in eukaryotic organisms involves a complicated interplay between a number of proteins required for licensing origins of replication and restricting the replication of the genome to only once per cell cycle [1,2,3,4,5]
Identification of Residues Putatively Required for Ring Dimerization—By analyzing the crystal structure of the dodecameric Methanothermobacter thermautotrophicus (MthMCM) N-terminal domain [15] (Protein Data Bank: 1LTL), we identified arginine residues (Arg-137, Arg160) on either side of the zinc finger motif with the potential to form hydrogen bonds across the ring-ring interface (Fig. 1A)
A number of electron microscopy studies on MthMCM have highlighted a remarkable degree of polymorphism, raising the question of the physiological significance of the multiple conformations observed in vitro and their role in the mechanism of helicase activity

Summary

EXPERIMENTAL PROCEDURES

Cloning and Mutagenesis—pJC025 [19] was used as a template for site-directed mutagenesis. Reaction mix containing 50 mM Hepes, pH 7.6, 1 mM dithiothreitol, 0.1 mg/ml bovine serum albumin (HDB), 7 mM MgCl2, 1 nM labeled template, increasing amounts of protein (0 –300 nM monomer), and 4 mM ATP was prepared on ice. Reactions were processed as described [13]. Single images were initially band pass-filtered with a low frequency cutoff of 150 Å and a high frequency cutoff of 15 Å After centering to their rotationally averaged total sum, the ring-shaped particle images were treated with multivariate statistical analysis to determine the main symmetry components contained (multivariate statistical analysis (MSA) symmetry analysis [34]). Single particle reconstitution for the RRAA mutant was carried out by using 1600 particles and by applying 7-fold symmetry based on the result of MSA symmetry analysis. The structure was refined to a resolution of 22 Å, according to the one-half bit criterion

RESULTS

A DNA Binding Phenotype Is Associated with R137A—The

DISCUSSION