Functions of the ATP hydrolysis subunits (RecB and RecD) in the nuclease reactions catalyzed by the RecBCD enzyme from Escherichia coli

Abstract

The RecBCD enzyme from Escherichia coli is an ATP-dependent nuclease and helicase. Two of its subunits, the RecB and RecD proteins, are DNA-dependent ATPases. We have purified RecB and RecD proteins with mutations in their consensus ATP binding sites to study the functions of these subunits in the ATP-dependent nuclease activities of RecBCD. Reconstituted heterotrimeric enzymes were prepared by mixing wild-type RecB or RecB-K29Q mutant protein (RecB∗) with purified RecC protein, and with a histidine-tagged wild-type RecD (hD) or mutant hRecD-K177Q (hD∗) protein. RecBCD and all four reconstituted enzymes (wild-type, two single mutants, and the double mutant) cleave a single-stranded DNA oligomer substrate (25-mer) in the absence of ATP at rates of 0.03 to 0.06 min −1. The nuclease reaction catalyzed by RecB∗ChD∗ is not stimulated significantly by ATP, while the reactions catalyzed by RecBCD, RecBChD, RecBChD∗, and RecB∗ChD are 300 to 3000 fold faster in the presence of 0.5 mM ATP. RecB∗ChD∗ also has very low ATP hydrolysis activity (∼10 3-fold less than RecBCD), as do the individual mutant RecB∗ and hRecD∗ proteins (∼100-fold less than RecB or hRecD). The products from the ATP-stimulated nuclease reaction with the oligomer substrate suggest a mechanism where two DNA molecules bind to the enzyme in opposite orientations and are cleaved by the nuclease active site. Cleavage towards the 3′-end of one oligomer (observed with RecBChD∗) depends on the wild-type RecB subunit, while RecD-dependent cleavage (observed with RecB∗ChD) occurs towards the 5′-end of the second bound oligomer.