ABSTRACT
Pulsed field gel electrophoresis (PFGE) is a quick and reliable procedure to resolve DNA molecules larger than 30 kb by applying an electric field that periodically changes direction. This technique can be used to estimate genome size of a microorganism, to reveal if a genome is circular or linear, to indicate the presence of megaplasmids, and to show if a strain contains only one or more chromosomes.
Subject(s)
Archaea/genetics , Bacteria/genetics , Electrophoresis, Gel, Pulsed-Field/methods , Genome Size , Genome, Archaeal , Genome, Bacterial , Base Composition , Chromosomes, Archaeal/chemistry , Chromosomes, Bacterial/chemistry , DNA, Archaeal/chemistry , DNA, Archaeal/genetics , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , Electrophoresis, Agar Gel , Physical Chromosome Mapping , Plasmids/chemistry , Plasmids/geneticsABSTRACT
Deregulation of mini-chromosome maintenance (MCM) proteins is associated with genomic instability and cancer. MCM complexes are recruited to replication origins for genome duplication. Paradoxically, MCM proteins are in excess than the number of origins and are associated with chromatin regions away from the origins during G1 and S phases. Here, we report an unusually wide left-handed filament structure for an archaeal MCM, as determined by X-ray and electron microscopy. The crystal structure reveals that an α-helix bundle formed between two neighboring subunits plays a critical role in filament formation. The filament has a remarkably strong electro-positive surface spiraling along the inner filament channel for DNA binding. We show that this MCM filament binding to DNA causes dramatic DNA topology change. This newly identified function of MCM to change DNA topology may imply a wider functional role for MCM in DNA metabolisms beyond helicase function. Finally, using yeast genetics, we show that the inter-subunit interactions, important for MCM filament formation, play a role for cell growth and survival.
Subject(s)
Archaeal Proteins/chemistry , DNA-Binding Proteins/chemistry , Sulfolobus solfataricus , Archaeal Proteins/ultrastructure , Binding Sites , Chromosomes, Archaeal/chemistry , Crystallography, X-Ray , DNA, Archaeal/chemistry , DNA, Superhelical/chemistry , DNA-Binding Proteins/ultrastructure , Electron Microscope Tomography , Models, Molecular , Protein Binding , Protein Interaction Domains and Motifs , Protein Multimerization , Protein Structure, Quaternary , Protein Structure, SecondaryABSTRACT
The primary candidate for the eukaryotic replicative helicase is the MCM2-7 complex, a hetero-oligomer formed by six AAA+ paralogous polypeptides. A simplified model for structure-function studies is the homo-oligomeric orthologue from the archaeon Methanothermobacter thermoautotrophicus. The crystal structure of the DNA-interacting N-terminal domain of this homo-oligomer revealed a double hexamer in a head-to-head configuration; single-particle electron microscopy studies have shown that the full-length protein complex can form both single and double rings, in which each ring can consist of a cyclical arrangement of six or seven subunits. Using single-particle techniques and especially multivariate statistical symmetry analysis, we have assessed the changes in stoichiometry that the complex undergoes when treated with various nucleotide analogues or when binding a double-stranded DNA fragment. We found that the binding of nucleotides or of double-stranded DNA leads to the preferred formation of double-ring structures. Specifically, the protein complex is present as a double heptamer when treated with a nucleotide analogue, but it is rather found as a double hexamer when complexed with double-stranded DNA. The possible physiological role of the various stoichiometries of the complex is discussed in the light of the proposed mechanisms of helicase activity.
Subject(s)
Archaeal Proteins/metabolism , Chromosomes, Archaeal/metabolism , DNA Helicases/chemistry , DNA Helicases/metabolism , Adenosine Diphosphate/pharmacology , Archaeal Proteins/chemistry , Archaeal Proteins/isolation & purification , Archaeal Proteins/ultrastructure , Chromosomes, Archaeal/chemistry , DNA/metabolism , DNA Helicases/genetics , Escherichia coli/genetics , Methanobacteriaceae/enzymology , Models, Biological , Nucleotides/metabolism , Protein Binding , Protein Conformation , Protein Structure, Tertiary , Substrate Specificity , Transformation, GeneticABSTRACT
Methanobacterium thermoautotrophicum minichromosome maintenance complex (mtMCM), a cellular replicative helicase, is a useful model for the more complex eukaryotic MCMs. Biochemical and crystallographic evidence indicates that mtMCM assembles as a double hexamer (dHex), but previous electron microscopy studies reported only the presence of single heptamers or single hexamers (Pape, T., Meka, H., Chen, S., Vicentini, G., Van Heel, M., and Onesti, S. (2003) EMBO Rep. 4, 1079-1083; Yu, X., VanLoock, M. S., Poplawski, A., Kelman, Z., Xiang, T., Tye, B. K., and Egelman, E. H. (2002) EMBO Rep. 3, 792-797). Here we present the first three-dimensional electron microscopy reconstruction of the full-length mtMCM dHex in which two hexamers contact each other via the structurally well defined N-terminal domains. The dHex has obvious side openings that resemble the side channels of LTag (large T antigen). 6-fold and 7-fold rings were observed in the same mtMCM preparation, but we determined that assembly as a double ring favors 6-fold structures. Additionally, open rings were also detected, which suggests a direct mtMCM loading mechanism onto DNA.
