118 Nucleic Acids Research, 2008, Vol. 36, No. 1 the subunit interactions, by tethering ends, promote aggregation of diﬀerent ssDNA–RecN–ATP–Mg2+ com- plexes to form a rosette-like structure. This is consistent with the observation that addition of a large excess of unlabelled ssDNA (500-fold) and ATP–Mg2+ to a pre-formed ssDNA–RecN complex did not reduce the formation of large nucleoprotein aggregates (25). The RecN dynamic architecture and end-binding speciﬁcity inﬂuenced by ATP–Mg2+ (25, this work) resemble the binding speciﬁcity of the HsRad50–Mre11–ATP– Mg2+ or the HsRad50–Mre11–Nbs1–ATP–Mg2+ com- plexes and also show some clear diﬀerences (25, this work, 36–38). Indeed, the Rad50–Mre11 complex, which exhibits ATP-independent dsDNA binding and such binding activity is stimulated by ATP, does not seem to bind ssDNA (39–41). The pre-formed ssDNA–RecN–ATP–Mg2 complexes are insensitive to the addition of SsbA, arguing that RecN should bind ssDNA prior or concomitantly with SsbA. Independent of the order of addition of sub- stoichiometric concentrations of RecA, the pre-formed ssDNA–RecN–ATP–Mg2 (CII and CIII) complexes were disassembled or not formed. However, RecA failed to dislodge RecN from the CI complex. AFM showed that when homologous circular DNA and sub-saturating RecA concentration were incubated with pre-formed ssDNA–RecN–ATP–Mg2 (CII–CIII) complexes RecA- promoted DNA strand invasion with more than 12-fold higher eﬃciency when compared to the yield of joint molecules in the absence of RecN. Under this condition, RecA should be located at the joint site of the DNA molecules, because RecA–ATPgS–Mg2+ is located at this site. We propose that RecA interacts with and promotes a conformational change of RecN, because the pre-formed ssDNA–RecN–ADP–Mg2+ complex, which is resistant to nearly saturating concentrations of chaotropic agents (25), can be disassembled by sub-stoichiometric concentrations of RecA. The in vivo roles of RecN Genetic and cytological evidence suggests that: (i) RecN and Ku/SbcC deﬁne diﬀerent avenues for DSB repair (17,18,21); (ii) RecN, which plays an important role in the repair of DSBs, acts prior to and after end-processing (17,20); (iii) RecN co-localizes with RecO, RecR and RecA on the nucleoid in a majority of cells after induction of DSBs (17,21) and (iv) in the wild type background the SsbC foci mostly co-localized with the DNA polymerase complex al late times (120 min after induction of a DSB) (18). It is likely that RecN, which is ubiquitous in bacteria, provides a paradigm for the overall eﬀect of the eukaryotic MRN(X) complex in recombination in vivo (42). Do the in vivo foci of RecN and rosette-like structures formed in vitro relate directly to these repair/recombina- tion events? The formation of RecN foci should be a multi-stepwise process, and in the bases of previous and the results presented, we proposed the following model (Figure 6). The details of this model are speculative at this point, but it is appealing because it incorporates the cytological, biochemical and structural infor- mation available: in step 1, RecN (Figure 6A) or RecN– ATP–Mg2+ can bind to ssDNA forming CI at gaps or CII–CIII complexes at ssDNA ends (25) as early as 15 min after DSB induction (17). In step 2, the resection of the 50-ends of duplex DNA by RecJ in concert with a RecQ- like enzyme (RecQ and/or RecS) or the AddAB helicase– nuclease complex enhances the formation of 1 or 2 discrete RecN foci (Figure 6B). At this stage DNA end-processing, directly or indirectly, might repress the NHEJ activity and favour HR by error-free repair of DSBs (19). In the absence of both AddAB and RecJ functions many RecN foci, and absence of RecA threads were observed (20,21). In step 3, the ssDNA–RecN–ATP–Mg2+ complex protects the 30-OH terminus of a DNA molecule (25), and favours ssDNA–RecN–ATP–Mg2+ inter-complex association, thereby increases the local concentration of ssDNA ends (Figure 6C). It is likely that RecN facilitates this process by tethering the ssDNA ends that are visualized by AFM as rosette-like structures (Figure 4), by EMSA as CII- and CIII-type complexes (25; Figure 5A) and as discrete RecN focus on the nucleoids in a majority of cells (17). We predict a dynamic behaviour of the ssDNA–RecN complexes that must be able to roam the nucleoid space in search for a discrete ssDNA–RecN– ATP–Mg2+ complex (RecN focus). In step 4, SsbA binds to ssDNA extensions generated by DNA end-processing (Figure 6C). The RecN rosette-like structures (CII- and CIII-type complexes) formed in step 3 cannot be disassembled by the addition of a large excess of the SsbA protein (Figure 5A), and SsbA (independent of the other of addition) partially inhibits the RecA ATPase activity (Carrasco et al., unpublished data). In step 5, RecA-mediators displace SsbA from ssDNA and promote RecA loading onto ssDNA. In the absence of SsbA, RecA promotes the dislodging of the large RecN networks resulting in a large concentration of ssDNA ends (Figure 6D). Lastly, RecA polymerizes on the clustered ssDNA molecules to make pre-synaptic nucleoprotein ﬁlaments. The diﬀerent RecA nucleoprotein ﬁlaments can then make single or multiple search for homology on the sister chromosome or sister strand, thus facilitating the search for genome-wide homology. This is in agree- ment with the observation that: (i) RecA forms foci, that co-localize with RecN, and threads or ﬁlamental structures upon DNA damage (20,43) and (ii) RecN augments RecA-mediated DNA strand invasion (Figure 5C). Both the eukaryotic MRN(X) complex and bacterial RecN in concert with DNA end-processing functions are able to detect DNA damage, play a structural role by bridging and tethering DNA ends and may provide the structural basis for the discrimination between the diﬀerent avenues of DNA repair (HR versus NHEJ) (17,18,36,37,42,this work,44,45). SUPPLEMENTARY DATA Supplementary Data are available at NAR Online.