structures were insensitive to even 1 mM SsbA (data not shown). Addition of sub-saturating amounts of RecA (40 nM) did not disrupt CI complexes, but in the presence of NT blocked the assembly of the slower migrating RecN–ssDNA CII and CIII complexes (5Ai, lanes 8–10) or did promote their disassembly (Figure 5Aii, lanes 8–10). Under these experimental conditions, RecA protein forms meta-stable complexes with ssDNA because complexes attributed only to ssDNA–RecA–ATP–Mg2+ were not observed (5Aii, lane 12). The absence of CII- and CIII-type complexes cannot be attributed to RecA- mediated hydrolysis of the ATP–Mg2+ because RecA promotes the disassembly of CII and CIII complexes even in the presence of ADP. AFM visualization of RecA interactions with RecN–DNA complexes In previous studies, it was shown that RecA-promoted pairing of homologous circular dsDNA with linear duplex with single-stranded extensions involved co-aggregation into large nucleoprotein networks (32,33; Figure 5B). To further characterize RecA interactions with ssDNA– RecN complexes, RecA-mediated DNA strand invasion complexes were visualized by AFM. In the absence of RecN, the linear duplex DNA with short ssDNA tails was pre-incubated with ATP–Mg2+, and then with aliquots of homologous circular dsDNA plus sub-saturating amounts of RecA. RecA (40 nM) catalysed DNA strand invasion was observed in 2% of total DNA molecules in a 10 min incubation time after deproteinization, but accumulation of joint molecules was not observed (<0.5%) if RecA was omitted. RecN bound to the Exo- generated single-stranded extensions of dsDNA molecules was pre-incubated with ATP–Mg2+, and then homolo- gous circular dsDNA plus sub-saturating amounts of RecA (40 nM) were added. RecA-mediated DNA strand invasion was observed in 40% of total DNA molecules in a 10 min incubation time (Figure 5C). The large rosette-like complexes seen with RecN-ATP- Mg2+ bound to the ssDNA tails of duplex molecules (Figure 4) were not detected in the presence of RecA. Although, RecA–DNA joint molecules were observed (Figure 5C, compare with the aggregates obtained if Nucleic Acids Research, 2008, Vol. 36, No. 1 117 no RecN was added Figure 5B). These observations are consistent with RecA-promoting disassembly of the large CII- and CIII-type RecN–ssDNA complexes, and catalysing DNA strand invasion into a circular homol- ogous dsDNA molecule in the presence of pre-formed ssDNA–RecN–ATP–Mg2+ complexes (Figure 5C). RecN protein fails to promote joint molecule formation in the absence of RecA protein (data not shown). To identify which protein was bound to the joint molecule (Figure 5C), we took advantage of the fact that ATPgS–Mg2+ binding to RecA–ssDNA complexes pro- motes nucleoprotein ﬁlament formation (reviewed by 34,35). Pre-formed ssDNA–RecN–ATP–Mg2+ complexes were incubated with ATPgS–Mg2+ followed by addition of homologous circular dsDNA plus RecA. In the presence of ATPgS–Mg2+, the large rosette-like com- plexes seen with RecN bound to ssDNA tails of duplex molecules were not detected, although long nucleoprotein ﬁlaments of RecA protein were formed at the joint molecule (Figure 5D), consistent with RecA being located at the linear-circular DNA interaction. DISCUSSION Different modes of RecN binding to ssDNA Our AFM studies demonstrate that in the absence of ATP–Mg2+ RecN binds to ssDNA. This binding results in the formation of discrete ‘blobs’ on ssDNA. We did not observe preferential binding to ssDNA as compared to ssDNA within duplex DNA regions. However, in the presence of ATP–Mg2+ the 30-OH DNA ends were strictly required for the formation of large ssDNA–RecN–ATP– Mg2+ (CII and CIII) complexes (25, Figure 5A). We observed that RecN forms also ‘blobs’ on nicked sites on duplex DNA, although, by EMSA experiments it was shown that in the absence of ATP–Mg2+ RecN forms stable complexes with ssDNA segments larger than 15-nt in length (25), thus we propose that RecN might interact with shorter ssDNA regions (e.g. nicked DNA) in a meta- stable manner. In the presence of ATP–Mg2+, the RecN–ssDNA complexes assembled to form the large CII and CIII complexes (Figure 4). It is likely that RecN bound to ssDNA translocates towards the ssDNA ends and Figure 5. RecA interaction with ssDNA–RecN–ATP–Mg2+ networks. (A) [g-32P] ssDNA60 (60 nM in nt) and RecN (10 nM) were pre-incubated for 10 min at 378C in buﬀer C. (Ai) SsbA (100 nM, lanes 5–7) or RecA (40 nM, lanes 8–10) protein was added to pre-formed RecN–ssDNA complexes and incubated for 10 min at 378C, then 1 mM ADP (lanes 6 and 9) or 1 mM ATP (lanes 7 and 10) or no nucleotide cofactor (lanes 5 and 8) was added, and incubated for 10 min at 378C. (Aii) 1 mM ADP (lanes 3, 6 and 9) or 1 mM ATP (lanes 4, 7 and 10) or no nucleotide cofactor (lanes 2, 5 and 8) was added to pre-formed RecN–ssDNA complexes, and then 100 nM SsbA (lanes 5–7) or 40 nM RecA (lanes 8–10) was added and incubated for 10 min at 378C. The protein(s)–ssDNA–NT–Mg2+ complexes were fractionated and visualized. In the absence of RecN, [g-32P] ssDNA60 (60 nM in nt) was incubated with RecA (40 nM, lanes 11 and 12) or SsbA (0.15 nM, lanes 13 and 14) in presence or absence of ATP. FD, free DNA; CI, complex I; CII, complex II; CIII, complex III; NT, presence of ADP or ATP; , denotes presence or absence of indicated factor. The SsbA–ssDNA complex (SsbA) is indicated. (B) RecA–DNA aggregates in the absence of RecN. Linear DNA (150 nM) with short ssDNA tails and 40 nM RecA were incubated for 10 min at 378C, in the presence of 1 mM ATP at 378C. Homologous circular plasmid DNA (150 nM) was added and incubated for another 10 min at 378C, and deposited on freshly cut mica. Scale bar ¼ 250 nm. (C) RecA-promoted DNA strand invasion in presence of RecN and ATP. Linear DNA (150 nM) with short ssDNA tails and 10 nM RecN were pre-incubated for 10 min at 378C, then 1 mM ATP was added and incubated for 10 min at 378C. Homologous circular plasmid DNA (150 nM) and RecA protein (40 nM) were added to the pre-formed ssDNA–RecN–ATP–Mg2+ complex, incubated for another 10 min at 378C, and deposited on freshly cut mica. Scale bar ¼ 500 nm (inset picture, bar ¼ 100 nm). (D) RecA-promoted DNA strand invasion in presence of RecN and ATPgS. Linear DNA (150 nM) with short ssDNA tails and RecN (10 nM) were pre-incubated for 10 min at 378C, then 0.1 mM ATPgS was added and incubated for 10 min at 378C. Homologous circular plasmid DNA (150 nM) and RecA protein (40 nM) were added to the pre-formed ssDNA–RecN–ATPgS–Mg2+ complex, incubated for another 10 min at 378C and deposited on freshly cut mica. White arrowheads show nucleoprotein ﬁlaments of RecA and DNA. Scale bar ¼ 100 nm.