PMID:8387820
| Citation |
Korangy, F and Julin, DA (1993) Kinetics and processivity of ATP hydrolysis and DNA unwinding by the RecBC enzyme from Escherichia coli. Biochemistry 32:4873-80 |
|---|---|
| Abstract |
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. |
| Links | |
| Keywords |
Adenosine Triphosphate/metabolism; DNA Helicases/drug effects; DNA Helicases/genetics; DNA Helicases/metabolism; DNA-Binding Proteins/metabolism; Escherichia coli/enzymology; Escherichia coli Proteins; Exodeoxyribonuclease V; Exodeoxyribonucleases/drug effects; Exodeoxyribonucleases/genetics; Exodeoxyribonucleases/metabolism; Heparin/pharmacology; Hydrolysis/drug effects; Models, Genetic; Nucleic Acid Conformation; Structure-Activity Relationship |
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