Nucleic Acids Research, 2008, Vol. 36, No. 1 111 Figure 1. Secondary structure prediction and motifs of B. subtilis SMC-like proteins. The secondary structure B. subtilis SMC-like proteins [SMC, RecN, SbcC (also termed YirY) and SbcC2 (YhaN)] and its comparison with E. coli SbcC (EcoSbcC) and human Rad50 (HsRad50) were predicted using the COILS program (www.ch.embnet.org/software/COILS_form.html). The Walker A and B motifs and the CXXC and GGXXXGG motifs necessary for dimerization of Rad50, SbcC, EcoSbcC and SMC proteins, respectively, are indicated. The putative dimerization (Leu rich) motif of RecN is also indicated. Abbreviation: aa, aminoacids. establishes a discrete DNA repair centre (RC) (17,20). Recombination proteins localize diﬀerentially to a RecN-induced RC: RecO, RecR and RecA are recruited 30–45 min after induction of DSBs, RecF (60 min) and RecU (120 min) are recruited at a RecN-directed RC at later stages, whereas others [e.g. AddAB (analogue of Escherichia coli RecBCD), RecG, RecS, DNA polymerase I] do not form discernible cytological foci (17,20,21). In the absence of end-processing functions (addAB recJ strain) two to three (rather than one) RecN foci are formed, whereas RecA assembly at the RC is not observed (21). These observations suggest that RecN acts: (i) upstream of end-processing enzymes, perhaps as an early detector of DSBs, and (ii) after end-processing because the activity of the AddAB and/or RecJ exonu- cleases are required for the formation of one discrete RecN RC, and for RecA assembly at a RecN-directed RC (17,20,21). Furthermore, RecF failed to form foci in the absence of RecO, arguing that RecF, which shares structural conservation with the head domain of Rad50 (15), might work after RecA loading onto ssDNA. During natural genetic recombination, the DNA uptake proteins localize to a cell pole (22), and RecN protein oscillates between the poles of competent B. subtilis cells (23). The internalized ssDNA stops the RecN oscillations, and then threads of RecA are seen to emanate from the pole (23). It is likely that RecN interacts in vivo with incoming ssDNA molecules and thereby is localized at the pole containing the natural competence machinery. RecN also possesses biochemical activities associated with the eukaryotic MRN(X) complex (6,7,12,24). The globular N- and C-terminal regions of RecN should interact to form the active ATPase (25). In vitro RecN is a ssDNA-dependent ATPase and shows signiﬁcant ATP-independent DNA binding. However, in the pres- ence of a nucleotide cofactor (ATP or ADP), RecN binds speciﬁcally to 30-OH termini forming large RecN–ssDNA networks that are not disrupted in the presence of an excess of ssDNA molecules (25). Here, we report the visualization of dynamic complexes formed by RecN with ssDNA or duplex molecules containing internal ssDNA regions. Atomic force microscopy (AFM) has conﬁrmed that RecN binds to ssDNA regions and, in the presence of ATP, the interactions between individual ssDNA–RecN complexes favour inter-complex association and result in large nucleoprotein complexes. The pre-formed ssDNA– RecN–ATP–Mg2+ aggregates are insensitive to the addition of SsbA (counterpart of E. coli SSB or eukaryotic RPA protein), arguing that RecN should bind ssDNA prior or concomitantly with SsbA. Independent of the order of addition, RecA promotes the disassembly of the pre-formed ssDNA–RecN–ATP–Mg2+ complexes and promotes DNA strand exchange without the forma- tion of large RecA–DNA networks. We propose that RecN-promoted RCs facilitate RecA-promoted genome-wide homology search of the RecA pre-synaptic ﬁlaments. MATERIALS AND METHODS Enzymes reagents and protein purifications RecN, RecA and SsbA proteins were puriﬁed as described (17,26, Carrasco et al., unpublished data). The molar extinction coeﬃcient for RecN, RecA and SsbA were calculated to be 30 600, 15 200, 11 400/M/cm at 280 nm as previously described (26). Protein concentrations were determined using the above molar extinction coeﬃcients. RecN and RecA are expressed as moles of protein monomers and SsbA as tetramers. Alkali lysis puriﬁed pBluescript DNA was linearized by EcoRI (NEB) digestion, and CsCl-puriﬁed pBluescript DNA was linearized by EcoRV (NEB) digestion. The 60-nt ssDNA molecule (ssDNA60) used has been pre- viously described (25). DNA concentrations were estab- lished using molar extinction coeﬃcients using 8780 and 6500/M/cm at 260 nm for ssDNA and dsDNA, respec- tively. Unless otherwise stated, DNA concentrations are given as moles of nucleotides. AFM Analysis Aliquots of protein solutions, diluted in buﬀer A (10 mM HEPES, pH 7.5, 2 mM MgCl2), were spotted onto a freshly cleaved mica substrate pre-treated with 10 mM