In vitro reconstitution of the late steps of genetic recombination in E. coli.
Purified proteins have been used to reconstitute an in vitro system for the medial-to-late stages of recombination in E. coli. In this system, RecA protein formed recombination intermediates that were processed by the actions of the RuvA, RuvB, and RuvC proteins. RuvAB was found to promote branch migration, to dissociate the RecA filament, and to modulate the orientation of cleavage of Holliday junction resolution by RuvC. Monoclonal antibodies directed against RuvA, RuvB, or RuvC inhibited resolution in the reconstituted system. Specific protein-protein interactions between the branch migration motor (RuvB) and the resolvase (RuvC) were also observed. These results provide evidence for coordinated action during the late stages of recombination, possibly involving the assembly of a RuvABC branch migration/resolution complex.
Eggleston
A K
AK
Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms, United Kingdom.
Mitchell
A H
AH
West
S C
SC
D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D004265:DNA Helicases; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D004706:Endodeoxyribonucleases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins; D008957:Models, Genetic; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D011938:Rec A Recombinases / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic; D013997:Time Factors
D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C061260:RuvB protein, Bacteria; C069806:ruvC protein, E coli; D011938:Rec A Recombinases; D004706:Endodeoxyribonucleases; C071059:Holliday junction DNA helicase, E coli; D004265:DNA Helicases