Kinetics and processivity of ATP hydrolysis and DNA unwinding by the RecBC enzyme from Escherichia coli

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.

DNA Helicase Activity of the RecD Protein from Deinococcus radiodurans

“…However, RecD is dispensable for unwinding, since RecBC enzyme is also a helicase (41,42), and the major effect of removing RecD from RecBCD is on the nuclease activity of RecBCD. Loss of the RecD subunit reduces the nuclease activity to very low levels that are not biologically relevant (43,44).…”

The RecD subunit of the Escherichia coli RecBCD enzyme inhibits RecA loading, homologous recombination, and DNA repair

“…This model is supported by the fact that recD mutants are recombination-proficient (11,23,29) and hyperre-combination-proficient in the absence of Chi sites (11), and they cluster recombination events at the ends of DNA in lambda replication-blocked crosses (30). Purified RecBC enzyme (i.e., lacking RecD) has a low affinity for dsDNA (unpublished data, see Table 5) ends but can unwind DNA (16,31,32) and facilitates loading of RecA protein at the 3Ј termini of DNA during unwinding (16). This is in contrast to RecBCD enzyme, which requires a Chi site for significant loading of RecA protein (6), joint molecule formation (7), and recombination (18).…”

A Single Mutation, RecBD1080A, Eliminates RecA Protein Loading but Not Chi Recognition by RecBCD Enzyme

“…The RecBC enzyme (without the RecD subunit) is a very processive helicase but has no significant nuclease activity (16,22,23). Recently, it was shown that the RecBC enzyme also coordinates the loading of RecA protein and that this loading is independent of .…”

“…Ϫ1 cm Ϫ1 at 280 nm, derived by adding the respective extinction coefficients for the individual subunits (23). No contaminating protein bands were detected when 1 g of protein was loaded on an SDS-polyacrylamide gel and stained with Coomassie Brilliant Blue.…”