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 k(cat) = 1600 min-1, K(m) = 8.1 muM, and k(cat)/K(m)(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.