Kinetics and processivity of ATP hydrolysis and DNA unwinding by the RecBC enzyme from Escherichia coli.
The RecB and RecC subunits of the RecBCD enzyme from Escherichia coli were purified from cells containing plasmids overproducing these proteins [Boehmer, P.E., & Emmerson, P.T. (1991) Gene 102, 1-6]. RecB hydrolyzes ATP in the presence of either single- or double-stranded DNA. RecC stimulates ATP hydrolysis by RecB, particularly with double-stranded DNA. The steady-state kinetic parameters for ATP hydrolysis by RecBC with double-stranded DNA are kcat = 1600 min-1, Km = 8.1 microM, and kcat/Km(ATP) = 1.97 x 10(8) M-1 min-1. The RecBC enzyme acts processively, as measured by the effect of heparin on ATP hydrolysis stimulated by double-stranded DNA. About 2400 ATP molecules are hydrolyzed per enzyme bound to the end of a DNA molecule, using DNA substrates of 6250 or 21,400 base pairs. The enzyme is capable of unwinding a 6250 base pair double-stranded DNA molecule, in the presence of the single-stranded DNA binding protein of Escherichia coli. The steady-state kinetic parameters and the processivity are close to those found previously for the RecBCD-K177Q enzyme, with a lysine-to-glutamine mutation in the consensus ATP binding sequence in the RecD subunit, and are reduced compared to the RecBCD holoenzyme [Korangy, F., & Julin, D. A. (1992) J. Biol. Chem. 267, 1733-1740]. The most salient difference between RecBC and RecBCD-K177Q is the nuclease activity. RecBCD-K177Q produces a significant amount of acid-soluble DNA fragments from double-stranded DNA, while RecBC does not, even though the DNA does become unwound.
Korangy
F
F
Department of Chemistry and Biochemistry, University of Maryland, College Park 20742.
Julin
D A
DA
D000255:Adenosine Triphosphate / Q000378:metabolism*; D004265:DNA Helicases / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D043211:Exodeoxyribonuclease V; D005090:Exodeoxyribonucleases / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D006493:Heparin / Q000494:pharmacology; D006868:Hydrolysis / Q000187:drug effects; D008957:Models, Genetic; D009690:Nucleic Acid Conformation; D013329:Structure-Activity Relationship
D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D000255:Adenosine Triphosphate; D006493:Heparin; D005090:Exodeoxyribonucleases; D043211:Exodeoxyribonuclease V; C468322:exodeoxyribonuclease V, E coli; D004265:DNA Helicases