110–120 Nucleic Acids Research, 2008, Vol. 36, No. 1 doi:10.1093/nar/gkm759 Published online 13 November 2007 Dynamic structures of Bacillus subtilis RecN–DNA complexes Humberto Sanchez1,2, Paula P. Cardenas 1 , Shige H. Yoshimura2, Kunio Takeyasu2 and Juan C. Alonso1,* 1Departamento de Biotecnologı´a Microbiana, Centro Nacional de Biotecnologı´a, CSIC, Campus Universidad Aut o´noma de Madrid, Darwin 3, Cantoblanco, 28049 Madrid, Spain and 2Laboratory of Plasma Membrane and Nuclear Signalling, Graduate School of Biostudies, Kyoto University, Sakyo-ku, Kitashirakawa-Oiwake-cho, Kyoto 606-8502, Japan Received July 20, 2007; Revised September 10, 2007; Accepted September 11, 2007 ABSTRACT Genetic and cytological evidences suggest that Bacillus subtilis RecN acts prior to and after end- processing of DNA double-strand ends via homo- logous recombination, appears to participate in the assembly of a DNA repair centre and interacts with incoming single-stranded (ss) DNA during natural transformation. We have determined the architecture of RecN–ssDNA complexes by atomic force microscopy (AFM). ATP induces changes in the architecture of the RecN–ssDNA complexes and stimulates inter-complex assembly, thereby increasing the local concentration of DNA ends. The large CII and CIII complexes formed are insensitive to SsbA (counterpart of Escherichia coli SSB or eukaryotic RPA protein) addition, but RecA induces dislodging of RecN from the over- hangs of duplex DNA molecules. Reciprocally, in the presence of RecN, RecA does not form large RecA–DNA networks. Based on these results, we hypothesize that in the presence of ATP, RecN tethers the 3’-ssDNA ends, and facilitates the access of RecA to the high local concentration of DNA ends. Then, the resulting RecA nucleoprotein filaments, on different ssDNA segments, might promote the simultaneous genome-wide homology search. INTRODUCTION All cells have systems dedicated to correct DNA damage, although if lesions are left unrepaired removal of single- strand (ss) DNA gaps by homologous recombination (HR) or removal of double-strand breaks (DSBs) via HR or non-homologous end joining (NHEJ) are the ultimate resource for the re-establishment of the replica- tion fork (1–7). Cytological experiments have documented the assembly of large nucleoprotein complexes that contain DNA- repair proteins in response to DNA damage (8–10). The mammalian Mre11–Rad50–Nbs1 (MRN) and budding yeast Mre11–Rad50–Xrs2 (MRX) complex [simpliﬁed as MRN(X) complex] are the early DSB sensors during mitotic recombination (5,8,10). It has been proposed that MRN(X) complexes act at the branching point upstream of both HR and NHEJ (11,12). The DNA ends are transiently stabilized, and there is a window of opportu- nity for NHEJ to occur. However, once the DNA ends are resected, the cell is committed to HR and NHEJ is no longer an option (6,7,11,12). The overall structure of the structural maintenance chromosome (SMC) protein Rad50 is evolutionary con- served from bacteria to humans. Bioinformatic studies suggest that bacterial SbcCs, SMC and RecN proteins share a similar modular organization with eukaryotic Rad50, a bipartite globular ATPase domain made up from the N- and C-termini of the protein separated by an extensive central region predicted to form a coiled-coil (13,14; Figure 1). Furthermore, the structural conserva- tion of RecF and Rad50 suggests a conserved mechanism of action (15). However, a clear spatial and temporal distinction between the localization of RecN, RecF, SMC and SbcC proteins was reported (16–18). In Bacillus subtilis two-ended DSBs are mainly repaired via HR, but with very low eﬃciency also by NHEJ (4,18). Genetic studies revealed that SbcC is epistatic with Ku (a key component of NHEJ) (19), but they are non- epistatic with RecN, suggesting that RecN and Ku/SbcC deﬁne diﬀerent avenues for DSB repair (18). RecN promotes the assembly of large nucleoprotein complexes that contain recombination proteins in response to DNA damages. In B. subtilis cells, 15 min after induction of a site-speciﬁc DSB or randomly introduced DSBs a massive reorganization of the nucleoid takes place, and RecN *To whom correspondence should be addressed. Tel: +34 91585 4546; Fax: +34 91585 4506; Email: jcalonso@cnb.uam.es 2007 The Author(s) This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.