Biochemical Characterization of a Mutant recBCD Enzyme, the recB 2109CD Enzyme, Which Lacks χ-specific, but Not Non-specific, Nuclease Activity

Abstract

RecBCD enzyme of Escherichia coli is a DNA helicase which also possesses ATP-dependent nuclease activities. We have purified a mutant recBCD enzyme, designated recB 2109CD enzyme, and have examined the nuclease activities of this protein in vitro to determine whether any alteration in these activities is responsible for the recombination-deficient phenotype of the recB2109 strain. The recB 2109CD enzyme possesses all of the non-specific nuclease activities (dsDNA exonuclease and ssDNA exo- and endonuclease) associated with wild-type recBCD enzyme although they are reduced ∼ 2 to 3-fold relative to the wild-type enzyme. The ATP -dependent dsDNA exonuclease activity of recB 2109CD enzyme requires significantly higher ATP concentrations for optimal activity when compared to the wild-type enzyme. The ATP-independent ssDNA endonuclease activity of the two enzymes is similar, but the ATP-stimulated ssDNA endonuclease and ATP-dependent ssDNA exonuclease activities of the mutant enzyme are reduced relative to those of wild-type recBCD enzyme. Despite its ability to degrade linear dsDNA non-specifically, recB 2109CD enzyme lacks sequence-specific nicking activity at χ sites, which are hotspots for genetic recombination. Since this interaction with χ significantly attenuates the non-specific dsDNA exonuclease activity of wild-type recBCD enzyme, these results suggest that the non-specific dsDNA exonuclease activity of the mutant enzyme cannot be attenuated, with the consequence that a DNA substrate which is suitable for recombination is not produced.