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Journal of Bacteriology, January 2006, p. 353-360, Vol. 188, No. 2
0021-9193/06/$08.00+0     doi:10.1128/JB.188.2.353-360.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Recruitment of Bacillus subtilis RecN to DNA Double-Strand Breaks in the Absence of DNA End Processing

Humberto Sanchez,1 Dawit Kidane,2,{dagger} M. Castillo Cozar,1 Peter L. Graumann,2,{dagger} and Juan C. Alonso1*

Department Microbial Biotechnology, Centro Nacional de Biotecnología, CSIC, C/Darwin 3, Campus Universidad Autónoma de Madrid, 28049 Madrid, Spain,1 Biochemie, Fachbereich Chemie, Hans-Meerwein-Straße, Philipps-Universität Marburg, 35032 Marburg, Germany2

Received 13 June 2005/ Accepted 19 October 2005

The recognition and processing of double-strand breaks (DSBs) to a 3' single-stranded DNA (ssDNA) overhang structure in Bacillus subtilis is poorly understood. Mutations in addA and addB or null mutations in recJ ({Delta}recJ), recQ ({Delta}recQ), or recS ({Delta}recS) genes, when present in otherwise-Rec+ cells, render cells moderately sensitive to the killing action of different DNA-damaging agents. Inactivation of a RecQ-like helicase ({Delta}recQ or {Delta}recS) in addAB cells showed an additive effect; however, when {Delta}recJ was combined with addAB, a strong synergistic effect was observed with a survival rate similar to that of {Delta}recA cells. RecF was nonepistatic with RecJ or AddAB. After induction of DSBs, RecN-yellow fluorescent protein (YFP) foci were formed in addAB {Delta}recJ cells. AddAB and RecJ were required for the formation of a single RecN focus, because in their absence multiple RecN-YFP foci accumulated within the cells. Green fluorescent protein-RecA failed to form filamentous structures (termed threads) in addAB {Delta}recJ cells. We propose that RecN is one of the first recombination proteins detected as a discrete focus in live cells in response to DSBs and that either AddAB or RecQ(S)-RecJ are required for the generation of a duplex with a 3'-ssDNA tail needed for filament formation of RecA.

* Corresponding author. Mailing address: Departamento de Biotecnología Microbiana, Centro Nacional de Biotecnología, C/Darwin 3, Campus Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain. Phone: (34) 91585 4546. Fax: (34) 915854506. E-mail: jcalonso{at}cnb.uam.es.

{dagger} Present address: Institut für Mikrobiologie, Universität Freiburg, Verfügungsgebäude, Stefan-Meier-Str. 17, 79104 Freiburg, Germany.

Journal of Bacteriology, January 2006, p. 353-360, Vol. 188, No. 2
0021-9193/06/$08.00+0     doi:10.1128/JB.188.2.353-360.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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