The mutant recBCD enzyme, recB²¹⁰⁹CD enzyme, has helicase activity but does not promote efficient joint molecule formation in vitro

The Escherichia coli recB²¹⁰⁹CD enzyme displays a defect in homologous recombination. In vitro, it possesses significant levels of non-specific nuclease activity but is deficient in χ-dependent nicking activity. To determine whether an alteration in helicase activity contributes further to its in vivo defect, the ability of recB²¹⁰⁹CD enzyme to unwind dsDNA was examined. The mutant enzyme is able to unwind DNA but has a k_cat which is one-third that of the wild-type enzyme, While the K_m for DNA ends of the wild-type and mutant enzymes at low NaCl concentration are essentially equivalent, the K_m for ATP of recB²¹⁰⁹CD enzyme is nearly six times greater. The processivity of unwinding (i.e. the average length of DNA unwound before recB²¹⁰⁹CD enzyme dissociates from the DNA substrate) at 1 mM-Mg²⁺ ion and 1 mM-ATP is approximately 13 kb/end, whereas that of wild-type recBCD enzyme is 30 kb/end. In an assay which requires the co-ordinate actions of the recBCD, recA, and SSB proteins, joint molecule formation in the presence of recB²¹⁰⁹CD enzyme is up to sixfold slower and proceeds to a lower extent than that mediated by the wild-type enzyme. We conclude that although the reduced helicase activity of the mutant recBCD enzyme may contribute to its recombination deficiency, its defect in the χ-dependent attenuation of non-specific nuclease activity is primarily responsible for the recombination-deficiency of E. coli strains bearing the recB2109 mutation.