EFFICIENCY OF ATP HYDROLYSIS AND DNA UNWINDING BY THE RECBC ENZYME FROM ESCHERICHIA-COLI

We have measured the rates and efficiencies of DNA unwinding (the numb er of ATP molecules hydrolyzed per DNA base pair unwound) catalyzed by the RecBC, RecBCD-K177Q (a site-directed mutant in the putative ATP-b inding site in the RecD subunit), and RecBCD enzymes from Escherichia coil. The DNA unwinding rate was measured with a coupled assay in whic h unwound DNA is degraded by the combined action of the RecJ enzyme an d exonuclease I. The rates of DNA unwinding by the RecBC and RecBCD-K1 77Q enzymes are reduced by about 4-fold compared to the case of the Re cBCD enzyme. The efficiency of ATP hydrolysis was determined in two wa ys. First, it was calculated from the ratio of the ATP hydrolysis rate to the rate of DNA unwinding. in the second method, ATP hydrolysis wa s measured under conditions where all of the DNA substrate becomes com pletely unwound. The efficiency is the ratio of the total amount of AT P hydrolyzed to the amount of DNA substrate present in the reaction. T he average efficiencies measured kinetically and by the complete unwin ding experiment are as follows: 2.30 and 1.74 ATP/base pair (RecBCD en zyme); 1.44 and 1.28 (RecBC); and 1.20 and 1.07 (RecBCD-K177Q). The Re cBC and RecBCD-K177Q enzymes are therefore able to couple ATP hydrolys is to DNA unwinding at least as efficiently as the RecBCD holoenzyme. The lower ATP per base pair ratios found for RecBC and RecBCD-K177Q in dicate that the RecD subunit hydrolyzes ATP during DNA unwinding by th e RecBCD enzyme.