title: Reconstitution of the activities of the RecBCD holoenzyme of Escherichia coli from the purified subunits.
abstract: The Escherichia coli RecBCD holoenzyme and the individual constituent subunits have been purified from overproducing strains. The purified RecBCD holoenzyme has a native molecular mass of approximately 330 kDa, indicative of a heterotrimer subunit assembly. The RecB, RecC, and RecD subunits can associate in vitro to give nuclease, helicase, ATPase, and Chi-specific endonuclease activities which are indistinguishable from those of the RecBCD holoenzyme. At concentrations at which the reconstituted RecB + C + D enzyme is very active, none of the individual RecB, RecC, or RecD subunits have readily detectable activities of the holoenzyme, except RecB protein which had previously been shown to exhibit DNA-dependent ATPase activity (Hickson, I. D., Robson, C. N., Atkinson, K. E., Hutton, L., and Emmerson, P. T. (1985) J. Biol. Chem. 260, 1224-1229). At higher concentrations and with shorter DNA substrates reconstituted RecBC protein exhibits low levels of helicase and exonuclease activity.
authors.affiliation: Department of Biochemistry and Genetics, Medical School, University, Newcastle upon Tyne, United Kingdom.
authors.forename: C
authors.initials: C
authors.lastname: Masterson
authors.forename: P E
authors.initials: PE
authors.lastname: Boehmer
authors.forename: F
authors.initials: F
authors.lastname: McDonald
authors.forename: S
authors.initials: S
authors.lastname: Chaudhuri
authors.forename: I D
authors.initials: ID
authors.lastname: Hickson
authors.forename: P T
authors.initials: PT
authors.lastname: Emmerson
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D002853:Chromatography, Liquid; D004269:DNA, Bacterial / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D043211:Exodeoxyribonuclease V; D005090:Exodeoxyribonucleases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed
chemical_list: D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D000255:Adenosine Triphosphate; D005090:Exodeoxyribonucleases; D043211:Exodeoxyribonuclease V; C468322:exodeoxyribonuclease V, E coli