Single-molecule study of RuvAB-mediated Holliday-junction migration.
Branch migration of Holliday junctions is an important step of genetic recombination and DNA repair. In Escherichia coli, this process is driven by the RuvAB complex acting as a molecular motor. Using magnetic tweezers, we studied the RuvAB-directed migration of individual Holliday junctions formed between two approximately 6-kb DNA molecules of identical sequence, and we measured the migration rate at 37 degrees C and 1 mM ATP. We directly demonstrate that RuvAB is a highly processive DNA motor protein that is able to drive continuous and unidirectional branch migration of Holliday junctions at a well defined average speed over several kilobases through homologous sequences. We observed directional inversions of the migration at the DNA molecule boundaries leading to forth-and-back migration of the branch point and allowing us to measure the migration rate in the presence of negative or positive loads. The average migration rate at zero load was found to be approximately 43 bp/sec. Furthermore, the load dependence of the migration rate is small, within the force range of -3.4 pN (hindering force) to +3.4 pN (assisting force).
Laboratoire Pierre Aigrain, Unité Mixte de Recherche 8551, Ecole Normale Supérieure, 24 Rue Lhomond, 75005 Paris, France.
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Dawid
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Croquette
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Grigoriev
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Heslot
D001426:Bacterial Proteins / Q000378:metabolism / Q000502:physiology; D001696:Biomechanical Phenomena; D004265:DNA Helicases / Q000378:metabolism / Q000502:physiology*; D045566:DNA, Cruciform / Q000378:metabolism / Q000648:ultrastructure*; D004268:DNA-Binding Proteins / Q000378:metabolism / Q000502:physiology*; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D007700:Kinetics; D046911:Macromolecular Substances; D008280:Magnetics; D008722:Methods; D008863:Microspheres; D020409:Molecular Motor Proteins; D009038:Motion
D001426:Bacterial Proteins; D045566:DNA, Cruciform; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D046911:Macromolecular Substances; D020409:Molecular Motor Proteins; C061260:RuvB protein, Bacteria; C071059:Holliday junction DNA helicase, E coli; D004265:DNA Helicases