In bacteria, double-strand DNA break (DSB) repair involves an exonuclease/helicase (exo/hel) and a short regulatory DNA sequence (Chi) that attenuates exonuclease activity and stimulates DNA repair. Despite their key role in cell survival, these DSB repair components show surprisingly little conservation. The best-studied exo/hel, RecBCD of Escherichia coli, is composed of three subunits. In contrast, RexAB of Lactococcus lactis and exo/hel enzymes of other low-guanine-plus-cytosine branch gram-positive bacteria contain two subunits. We report that RexAB functions via a novel mechanism compared to that of the RecBCD model. Two potential nuclease motifs are present in RexAB compared with a single nuclease in RecBCD. Site-specific mutagenesis of the RexA nuclease motif abolished all nuclease activity. In contrast, the RexB nuclease motif mutants displayed strongly reduced nuclease activity but maintained Chi recognition and had a Chi-stimulated hyperrecombination phenotype. The distinct phenotypes resulting from RexA or RexB nuclease inactivation lead us to suggest that each of the identified active nuclease sites in RexAB is involved in the degradation of one DNA strand. In RecBCD, the single RecB nuclease degrades both DNA strands and is presumably positioned by
RecD. The presence of two nucleases would suggest that this RecD function is dispensable in RexAB.In bacteria, double-stranded DNA breaks (DSB) are frequent events that may be provoked, for example, by pauses in the replication fork (36, 43). Such genomic disruptions are lethal in the absence of DNA repair. In Escherichia coli DSB repair requires the activity of a large enzyme complex, known as RecBCD, that has ATP-dependent helicase and exonuclease activities (see reference 32 for a review). The enzyme degrades both strands, starting from the DNA break until it reaches an octanucleotide sequence, known as Chi, that attenuates degradation and stimulates recombination (44,46). The enzyme exhibits helicase activity and residual exonuclease activity with an altered polarity after Chi (4, 16); the remaining activity provides a single-stranded DNA substrate for recombination enzymes to mediate repair.Organization of the three-subunit exonuclease/helicase (exo/hel) RecBCD. Structure-functional studies of RecBCD have revealed some of the roles of each subunit. RecB seems to possess two key activities of the enzyme. The N-terminal 929 amino acids (out of 1,180 total) have confirmed ATPase and helicase activities (13,54