In vivo evidence for two active nuclease motifs in the double-strand breakrepair enzyme RexAB of Lactococcus lactis

In bacteria, double-strand DNA break (DSB) repair involves an exonuclease/h elicase (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 conserv ation. The best-studied exo/hel, RecBCD of Escherichia coli, is composed of three subunits, In contrast, RexAB of Lactococcus lactis and exo/hel enzym es of other low-guanine-plus-cytosine branch gram-positive bacteria contain two subunits, We report that RexAB functions via a novel mechanism compare d 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, t he RexB nuclease motif mutants displayed strongly reduced nuclease activity but maintained Chi recognition and had a Chi-stimulated hyper-recombinatio n phenotype, The distinct phenotypes resulting from RexA or RexB nuclease i nactivation 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 posit ioned by RecD. The presence of two nucleases would suggest that this RecD f unction is dispensable in RexAB.