The RuvABC resolvasome.
The RuvABC resolvasome of Escherichia coli catalyses the resolution of Holliday junctions that arise during genetic recombination and DNA repair. This process involves two key steps: branch migration, catalysed by the RuvB protein that is targeted to the Holliday junction by the structure specific RuvA protein, and resolution, which is catalysed by the RuvC endonuclease. We have quantified the interaction of the RuvA protein with synthetic Holliday junctions and have shown that the binding of the protein is highly structure-specific, and leads to the formation of a complex containing two tetramers of RuvA per Holliday junction. Our data are consistent with two tetramers of RuvA binding to the DNA recombination intermediate in a co-operative manner. Once formed this complex prevents the binding of RuvC to the Holliday junction. However, the formation of a RuvAC complex can be observed following sequential addition of the RuvC and RuvA proteins. Moreover, by examining the DNA recognition properties of a mutant RuvA protein (E55R, D56K) we show that the charge on the central pin is critical for directing the structure-specific binding by RuvA.
Dickman
Mark J
MJ
Transgenomic Research Laboratory, Krebs Institute, Department of Molecular Biology and Biotechnology, University of Sheffield, UK.
Ingleston
Stuart M
SM
Sedelnikova
Svetlana E
SE
Rafferty
John B
JB
Lloyd
Robert G
RG
Grasby
Jane A
JA
Hornby
David P
DP
D001426:Bacterial Proteins / Q000737:chemistry*; D001665:Binding Sites; D004247:DNA / Q000737:chemistry*; D004265:DNA Helicases; D004268:DNA-Binding Proteins / Q000737:chemistry*; D004706:Endodeoxyribonucleases / Q000737:chemistry*; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry*; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D011485:Protein Binding; D011995:Recombination, Genetic; D020349:Surface Plasmon Resonance; D013997:Time Factors
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C061260:RuvB protein, Bacteria; C069806:ruvC protein, E coli; D004247:DNA; D004706:Endodeoxyribonucleases; C071059:Holliday junction DNA helicase, E coli; D004265:DNA Helicases