The directionality of RuvAB-mediated branch migration: in vitro studies with three-armed junctions.
BACKGROUND
The Escherichia coli RuvA and RuvB proteins promote the branch migration of 4-way (Holliday) junctions during genetic recombination. The active complex is a tripartite structure in which RuvA protein is bound to the crossover and is sandwiched between two hexameric rings of RuvB. Branch migration requires ATP hydrolysis and occurs as the DNA passes through each RuvB ring.


RESULTS
In this work, we have investigated the mechanism by which RuvAB catalyses the branch migration of a three-armed (Y) junction. Using synthetic DNA structures, we observed the formation of DNA products, a partial duplex DNA molecule and a single-stranded oligonucleotide, indicative of a branch migration reaction that occurred with unique polarity. Analysis of the RuvAB-junction complex by DNase footprinting showed that RuvA bound asymmetrically to the junction and targeted a single hexameric RuvB ring to one arm of DNA.


CONCLUSIONS
Branch migration of a three-armed junction occurs in a unidirectional manner that is determined by the assembly of a single RuvB ring onto one arm of the DNA. The asymmetry of the complex and observed directionality of branch migration indicate that strand passage occurs as the DNA is pulled into the RuvB ring structure, a reaction likely to be coupled with DNA unwinding.
Hiom
K
K
Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms, Hertfordshire, UK.
Tsaneva
I R
IR
West
S C
SC
D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D018983:DNA Footprinting; D004265:DNA Helicases; D004269:DNA, Bacterial / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D003850:Deoxyribonuclease I; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009690:Nucleic Acid Conformation; D011487:Protein Conformation; D011995:Recombination, Genetic
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C061260:RuvB protein, Bacteria; D003850:Deoxyribonuclease I; C071059:Holliday junction DNA helicase, E coli; D004265:DNA Helicases