INTERACTIONS OF THE RECBCD ENZYME FROM ESCHERICHIA-COLI AND ITS SUBUNITS WITH DNA, ELUCIDATED FROM THE KINETICS OF ATP AND DNA HYDROLYSIS WITH OLIGOTHYMIDINE SUBSTRATES

Oligothymidines eight nucleotides or longer stimulate ATP hydrolysis b y the RecBC and RecBCD enzymes, and they are substrates for the ATP-st imulated nuclease activity of RecBCD. The steady-state kinetics of ATP hydrolysis by the RecBC enzyme are consistent with a single ATPase an d DNA binding site. Results with RecBCD and RecBCD-K177Q [an enzyme wi th a Lys-to-Gln mutation in the ATP binding motif of the RecD subunit [Korangy, F., & Julin, D. A. (1992) J. Biol. Chem. 267, 1727-1732]] in dicate that ATP hydrolysis by the RecB subunit is stimulated by pd(T)( 12) binding to a high-affinity site, while the RecD subunit hydrolyzes ATP stimulated by pd(T)(12) binding to a low-affinity site. The site which stimulates RecB has about 50-fold greater affinity for DNA in ei ther RecBCD or RecBCD-K177Q than does the corresponding site in RecBC. The rates of ATP hydrolysis observed for the RecBCD enzyme at low con centrations of pd(T)(12) are best explained by a mechanism where the e nzyme binds to the DNA and catalyzes multiple rounds of ATP hydrolysis before dissociating. Larger DNA molecules [pd(T)(25-30) and poly(dT)] are bound more tightly by RecBCD, are hydrolyzed more rapidly, and ar e much more effective in stimulating ATP hydrolysis than is pd(T)(12). The results at low ATP concentrations where the nuclease activity is minimal (5 mu M) suggest that ATP hydrolysis is stimulated by the DNA ends, but there is no evidence that the RecBCD enzyme moves along thes e DNA molecules in an ATP-dependent manner under these conditions.