F. Korangy et Da. Julin, KINETICS AND PROCESSIVITY OF ATP HYDROLYSIS AND DNA UNWINDING BY THE RECBC ENZYME FROM ESCHERICHIA-COLI, Biochemistry, 32(18), 1993, pp. 4873-4880
The RecB and RecC subunits of the RecBCD enzyme from Escherichia coli
were purified from cells containing plasmids overproducing these prote
ins [Boehmer, P. E., & Emmerson, P. T. (1991) Gene 102, 1-6]. RecB hyd
rolyzes ATP in the presence of either single- or double-stranded DNA.
RecC stimulates ATP hydrolysis by RecB, particularly with double-stran
ded DNA. The steady-state kinetic parameters for ATP hydrolysis by Rec
BC with double-stranded DNA are k(cat) = 1600 min-1, K(m) = 8.1 muM, a
nd k(cat)/K(m)(ATP) = 1.97 X 10(8) M-1 min-1. The RecBC enzyme acts pr
ocessively, as measured by the effect of heparin on ATP hydrolysis sti
mulated by double-stranded DNA. About 2400 ATP molecules are hydrolyze
d 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 625
0 base pair double-stranded DNA molecule, in the presence of the singl
e-stranded DNA binding protein of Escherichia coli. The steady-state k
inetic parameters and the processivity are close to those found previo
usly for the RecBCD-K177Q enzyme, with a lysine-to-glutamine mutation
in the consensus ATP binding sequence in the RecD subunit, and are red
uced compared to the RecBCD holoenzyme [Korangy, F., & Julin, D. A. (1
992) J. Biol. Chem. 267, 1733-1740]. The most salient difference betwe
en RecBC and RecBCD-K177Q is the nuclease activity. RecBCD-K177Q produ
ces a significant amount of acid-soluble DNA fragments from double-str
anded DNA, while RecBC does not, even though the DNA does become unwou
nd.