The 30-kDa C-terminal domain of the RecB protein is critical for the nuclease activity, but not the helicase activity, of the RecBCD enzyme from Escherichia coli

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Abstract
The RecBCD enzyme from Escherichia coli is an ATP-dependent helicase and an ATP-stimulated nuclease. The 3′ → 5′ exonuclease activity on double-stranded DNA is suppressed when the enzyme encounters a recombinational hot spot, called chi (χ). We have prepared a RecB deletion mutant (RecB 1–929 ) by using results of limited proteolysis experiments that indicated that the RecB subunit consists of two main domains. The RecB 1–929 protein, comprising the 100-kDa N-terminal domain of RecB, is an ATP-dependent helicase and a single-stranded DNA-dependent ATPase. Reconstitution of RecB 1–929 with RecC and RecD leads to processive unwinding of a linearized plasmid. However, the reconstituted RecB 1–929 CD enzyme has lost the single-strand endo- and exonuclease and the double-strand exonuclease activities of the RecBCD enzyme. These results show that the 30-kDa C-terminal domain of RecB has an important role in the nuclease activity of RecBCD. On the basis of these findings, we propose the RecB C-terminal domain swing model to explain RecBCD’s transformation from a 3′ → 5′ exonuclease to a helicase when it meets a χ site.