Nucleic Acids Research, 2008, Vol. 36, No. 1 115 Figure 4. AFM analysis of RecN bound to DNA in presence of ATP. Linear DNA (150 nM in nt) with short ssDNA tails and RecN (10 nM) were incubated for 10 min at 378C, then ATP (1 mM) was added and incubated for 20 min at 378C. (A) 3D image of one ‘rosette-like structure’ stressing the height of the protein ‘blob’. (B) Gallery of AFM images of the diﬀerent species. Colour represents height from 0 to 5 nm, dark to light. Scale bar ¼ 100 nm. These data suggest that AMP-PNP apparently does not suﬃciently promote the active form of RecN required for inter-complex assembly (rosette-like structures). We can envisage a dynamic behaviour of RecN, with a large number of small RecN aggregates at the end of the DNA specimens (Figure 4B), and then a small number of large RecN aggregates with diﬀerent DNA ends bound to it forming ‘rosette-like structures’ (Figure 4A). RecA promotes disassembly of the ssDNA–RecN– ATP–Mg2+ complexes In previous studies it was shown that RecA protein preferentially hydrolyses dATP than ATP, and supports an eﬃcient DNA strand exchange in presence of dATP when compared to ATP (30,31, unpublished data). Addition of a large excess of RecA (0.2 mM), under optimized conditions for RecA binding (presence of dATP–Mg2+), promoted the disassembly of the pre- formed ssDNA–RecN–dATP–Mg2+ (CII and CIII) com- plexes (25). However, the RecN–ssDNA complexes formed in the presence of dATP–Mg2+ (CII and CIII complexes) were not disrupted by the addition of a heterologous ssDNA-binding protein (bacteriophage SPP1 single- stranded binding protein) (25). To investigate the fate of the rosette-like structures further, EMSA experiments, under conditions optimized for RecN binding to ssDNA, were undertaken in the presence or absence of SsbA, RecA and/or 1 mM ATP or ADP. In the absence of a nucleotide, RecN bound to ssDNA to form a complex designated CI (Figure 5A, lane 2), but with ATP–Mg2+ or with ADP– Mg2+, slower migrating RecN–ssDNA (CII and CIII) complexes were formed (Figure 5A, lanes 3 and 4 versus 2; 25). In the presence of limiting SsbA concentration (0.15 nM), one discrete SsbA–ssDNA60 complex was observed (5Aii, lane 14). Addition of a large excess of SsbA (100 nM) to the pre-formed CI complex was followed by the binding of SsbA to ssDNA (SsbA– ssDNA complexes, Figure 5Ai, lanes 5). ADP or ATP (denoted as NT) addition to the above reaction did not enhance CII and CIII complexes formation (Figure 5Ai, lanes 6 and 7). However, addition of SsbA (100 nM) to pre-formed ssDNA–RecN–ATP–Mg2+ or ssDNA–RecN–ADP–Mg2+ complexes failed to disas- semble the CII and CIII complexes (Figure 5Aii, lanes 6 and 7). Furthermore, the ssDNA–RecN–ATP–Mg2+ or ssDNA–RecN–ADP–Mg2+ complexes or rosette-like