RUVB PROTEIN-MEDIATED ATP HYDROLYSIS - FUNCTIONAL ASYMMETRY IN THE RUVB HEXAMER

A survey of RuvB protein-mediated ATP hydrolysis yields the following observations. (1) The RuvB protein exhibits a DNA-independent ATPase a ctivity with a turnover number (based on a RuvB monomer) approaching 6 min(-1) and a K-m of 154 mu M. Single-stranded DNA and linear duplex DNA have small but significant effects on this activity. (2) At ATP co ncentrations near the K-m, the ATPase activity is attenuated after sim ilar to 60 turnovers/RuvB monomer. The attenuation does not reflect in hibition by ADP. Addition of ATP to 3 mM triggers an immediate resumpt ion of ATP hydrolysis. The attenuation is enhanced somewhat by ssDNA a nd reduced somewhat by linear dsDNA. (3) ATP hydrolysis is dramaticall y stimulated by circular dsDNA, reinforcing the notion that RuvB trans locates along the DNA in a reaction coupled to ATP hydrolysis. The k(c at) increases by at least 2-4-fold on circular duplexes depending on c onditions, and the inactivation of RuvB at ATP concentrations near the K-m does not occur. The ATPase activity on circular dsDNA also exhibi ts a partial substrate inhibition by ATP. (4) Optimal ATP hydrolysis r equires similar to 1 DNA circle/RuvB hexamer, suggesting that multiple RuvB hexamers on a circle have an inhibitory effect on the ATPase act ivity. (5) With or without any of these DNA cofactors, a burst of ATP hydrolysis is observed under pre-steady-state conditions equivalent to 1 ATP per 3-3.3 RuvB monomers (2 ATP/hexamer). The substrate inhibiti on and burst results suggest the presence of nonequivalent ATP hydroly tic sites in a RuvB hexamer. The attenuation of ATPase activity observ ed under some conditions may also be a manifestation of nonequivalent ATP hydrolytic sites.