Nucleic Acids Research, 2008, Vol. 36, No. 1 113 Figure 2. RecN binds short ssDNA tails in a duplex molecule. (A) Linear DNA (150 nM in nt) was incubated for 10 min at 378C and deposited on freshly cut mica. (B) Linear DNA (150 nM) treated with Exo under limiting conditions and then incubated for 10 min at 378C and deposited on freshly cut mica. White arrowheads highlight the presence of ssDNA tails in the linear plasmids. (C) Linear DNA (150 nM) with short ssDNA tails and RecN (10 nM) were incubated for 10 min at 378C and deposited on freshly cut mica. Pictures show representative individual molecules. The graph shows the multimerization state of DNA-bound RecN complex. The sizes of the RecN complex bound to the ends of the DNA substrate were plotted against the width of bare DNA in the same image. The line shows the linear ﬁtting of the data (y ¼ 4.97 + 0.35X; R ¼ 0.82). Scale bar ¼ 100 nm. DNA molecules (Supplementary Figure S1), arguing for the presence of internal ssDNA regions. Based on the sizes of the internal SsbA complexes, the regions of ssDNA are suﬃcient to accommodate a SsbA tetramer (29; Supplementary Figure S1). Localization of RecN binding by endonuclease nicking of DNA CsCl-puriﬁed plasmid DNA was linearized with EcoRV to obtain blunt-ended linear dsDNA molecules. DNA aliquots were exposed to Nt.AlwI, which introduced asymmetric site-speciﬁc nicks, and then RecN was added. AFM revealed RecN bound to <10% of control dsDNA molecules that had not been subjected to Nt.AlwI digestion, but to essentially all the Nt.AlwI digested DNA molecules. The asymmetry in the Nt.AlwI generated nicking sites, four on the plus strand and six on the minus strand (Figure 3B, top and bottom line, respec- tively), allowed us to landmark the relative position of the DNA ends. Measurements from the ends of the DNA molecules, conﬁrmed that RecN molecules were posi- tioned predominantly at the site where Nt.AlwI intro- duced ssDNA nicks (Figure 3A). RecN also bound to other sequences that have a propensity to form ssDNA bubbles (namely dA–dT rich regions associated with DNA unwinding elements linked to phage f1 and plasmid replication origins). Alternatively, RecN may also bind to dA–dT rich regions on dsDNA, although RecN did not seem to bind preferentially to the upstream dA–dT rich regions of the lac promoter (Figure 3B), suggesting that ssDNA rather than dA–dT rich region is the main target of RecN in the absence of ATP. This is consistent with the observations that RecN binds in vivo to take up ssDNA during natural transformation (see Introduction section), and RecN cannot bind duplex DNA lacking dA–dT rich pockets. ATP-dependent aggregation of RecN–ssDNA complexes It was shown in the previous section that in the absence of a nucleotide co-factor RecN has no strong preference for ssDNA ends over internal sites. Using EMSA, we found that in the presence of ATP, RecN assembles to form large networks with ssDNA molecule and requires 30-OH at the ends of the ssDNA molecules (25). To investigate the interaction of RecN protein with 30-ssDNA-tailed duplex DNA, 1 mM ATP was added to the pre-formed RecN–30-ssDNA-tailed complex, the reac- tion was incubated for 20 min at 378C, and observed by AFM. Figure 4 shows diﬀerent scenarios with diﬀerent