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Journal of Bacteriology, July 2001, p. 4071-4078, Vol. 183, No. 13
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.13.4071-4078.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

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

Andréa Quiberoni,1,dagger Indranil Biswas,1,Dagger Meriem El Karoui,1 Lahcen Rezaïki,1 Patrick Tailliez,2 and Alexandra Gruss1,*

Laboratoire de Génétique Appliquée1 and Collection CNRZ---URLGA,2 Institut National de la Recherche Agronomique, Domaine de Vilvert, 78352 Jouy en Josas, France

Received 16 January 2001/Accepted 11 April 2001

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.

* Corresponding author. Mailing address: Laboratoire de Génétique Appliquée, Institut National de la Recherche Agronomique, Domaine de Vilvert, 78352 Jouy en Josas, France. Phone: 33-1 34 65 21 68. Fax: 33-1 34 65 20 65. E-mail: gruss{at}biotec.jouy.inra.fr.

dagger Permanent address: Facultad de Ingenieria Quimica, Universidad Nacional del Litoral, Santiago del Estero 2829, 3000 Santa Fe, Argentina.

Dagger Permanent address: National Center for Cell Science, Ganeshkhind, Pune 411007, India.

Journal of Bacteriology, July 2001, p. 4071-4078, Vol. 183, No. 13
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.13.4071-4078.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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