In Vivo Evidence for Two Active Nuclease Motifs in the Double-Strand Break Repair Enzyme RexAB of Lactococcus lactis

Abstract

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

RecBCD Enzyme and the Repair of Double-Stranded DNA Breaks

“…on May 11, 2018 by guest http://mmbr.asm.org/ symmetric DNA cleavage and the presence of two nuclease domains in AddAB have led to the attractive hypothesis that each nuclease domain is exclusively responsible for the cleavage of one ssDNA strand (57,59,226,227). Experiments with the RexAB enzyme (a Lactococcus lactis AddAB homologue) confirmed that both nuclease domains are active in vivo (226).…”

“…Experiments with the RexAB enzyme (a Lactococcus lactis AddAB homologue) confirmed that both nuclease domains are active in vivo (226). Recent work on Bacillus subtilis AddAB demonstrated that each nuclease domain is dedicated to the cleavage of just one DNA strand (AddA cleaves the 3Ј strand, and AddB cleaves the 5Ј strand) and that the generation of Chi-specific fragments requires the AddA, but not the AddB, nuclease domain (323).…”

DNA Repair and Genome Maintenance in Bacillus subtilis

“…The N terminus of AddA is an SF1A family helicase, and the C-terminal domain is a RecB-type nuclease, which cuts the 3=-5= strand (319,338,470). AddB does not have helicase activity; the C terminus of AddB forms a RecB-like nuclease domain which cleaves the 5=-3= strand (319,470). An Fe-S cluster is present in AddB, and this region has been shown to bind DNA in the crystal structure and to stabilize the protein structure (338).…”

The Recombination Genes addAB Are Not Restricted to Gram-Positive Bacteria: Genetic Analysis of the Recombination Initiation Enzymes RecF and AddAB in Rhizobium etli

“…Although RecBCD and AddAB/RexAB perform a similar role, several differences are observed: (i) RecBCD is constituted by three subunits, while AddAB/RexAB have two; (ii) although the identity between AddA and RecB is low, both share conserved regions corresponding to helicase and nuclease motifs (25,27,48) (Fig. 1B); (iii) only one nuclease motif is present in the RecB subunit of RecBCD, while AddAB have two active nuclease motifs, one in each subunit (48) (Fig.…”

“…1B); (iii) only one nuclease motif is present in the RecB subunit of RecBCD, while AddAB have two active nuclease motifs, one in each subunit (48) (Fig. 1C); and (iv) RecBCD has two helicase activities performed by the RecB and RecD subunits, while in AddAB, only AddA has helicase activity (12,20,27,49,59).…”