112 Nucleic Acids Research, 2008, Vol. 36, No. 1 spermidine. After 2 min, at room temperature, the mica was washed several times with MilliQ water and dried under nitrogen. AFM observations were performed using a Nanoscope IIIa or IV microscope (Veeco) in air using Tapping Mode. The cantilever (OMCL-AC160TS-W2, Olympus) was 129 mm in length with a spring constant of 33–62 N/m. The scanning frequency was 2–3 Hz, and images were captured with the height mode in a 512 512 pixel format. The obtained images were plane-ﬁtted and ﬂattened by the computer program accompanying the imaging module. We have assumed that the RecN-active ATPase has a globular domain. Based on the molecular mass of RecN (576 residues long, 64.4 kDa), we estimated the mass of the ATPase head domain of RecN monomers to be 40 kDa taking into account the hydration component (see subsequently), and expected a volume of 75 nm3 when measuring by AFM. The interactions of the putative globular N- and C-terminal regions might extrude the intervening coiled-coil region as a tail (Figure 1). However, we did not observe such tails and the particle sizes were larger than expected for just the globular domains, suggesting that the central connecting region also contributed to the total particle volume (data not shown). In the geometrical analyses of AFM images of DNA and protein, the ‘tip eﬀect’ was removed by using the apparent size of DNA as a reference as described elsewhere (27,28). Brieﬂy, the apparent dimensions of the molecules obtained by AFM are dependent upon the radius of the tip curvature and are apparently larger than the real dimensions. The relationship among the width of the globular molecule in the AFM image (W ), the radius of the tip curvature (Rc) and the radius of the molecule (Rm) is given by W ¼ 4(RcRm)1/2. When two diﬀerent molecules with Rm1 and Rm2 radii are imaged with the same tip, the relationship between the W1 measured ¼ W2(Rm1/Rm2)1/(2W.1Since widths and the W2) diameter can be of a given dsDNA by molecule is known (2 nm), we used the established parameter in our AFM image calculations as W1. We could then calculate the radius of the particles (Rm2) from the apparent width of the DNA (W2). EMSA experiments Reaction mixtures that contained [g-32P]-ssDNA60 (60 nM in nt) and RecN (10 nM) were assembled in buﬀer B [50 mM Tris–HCl (pH 7.5), 50 mM NaCl, 2 mM MgCl2, 1 mM DTT, 2% PEG-6000] for 10 min at 378C, then the second protein [SsbA (100 nM), RecA (40 nM)] and 10 min later ADP or ATP (1 mM) was added and incubation continued for 10 min at 378C. In some studies, SsbA or RecA was added to pre-formed ssDNA–RecN–ATP/ ADP–Mg2+ complexes. The complexes formed were separated by polyacrylamide gel electrophoresis and visu- alized by phosphorimaging as previously described (25). DNA substrates and binding reactions EcoRI-cleaved pBluescript DNA (2986-bp) was partially digested with Exo (Invitrogen) to generate short 30-ssDNA tails (linear duplex DNA with short ssDNA tails). EcoRV-cleaved pBluescript was exposed to Nt.AlwI endonuclease (NEB) to obtain blunt-ended DNA mole- cules with internal single-strand nicks. Protein–DNA complexes for AFM visualization were assembled as described for EMSA (25) and were deposited and imaged as described earlier. Linear duplex DNA with short ssDNA tails (150 nM) and RecN (10 nM) were pre-incubated for 10 min at 378C in buﬀer C [50 mM Tris–HCl (pH 7.5), 50 mM NaCl, 10 mM magnesium acetate, 50 mg/ml bovine serum albu- min (BSA), 1 mM DTT], then ATP or ATPgS (1 mM) was added and incubation continued for 10 min at 378C. To pre-formed ssDNA–RecN–ATP–Mg2+ or ssDNA– RecN–ATPgS–Mg2+ complexes, homologous circular plasmid DNA (150 nM) and RecA (40 nM) protein were added, and after incubation for 10 min at 378C the complexes were spotted on freshly cut mica. Linear duplex DNA with short ssDNA tails (150 nM), RecA (40 nM) and 1 mM ATP were pre-incubated for 10 min at 378C in buﬀer C and then homologous circular plasmid DNA (150 nM) was added, and after incubation for 10 min at 378C the complexes were spotted on freshly cut mica. RESULTS AFM visualization and quantification of RecN binding to ssDNA We were unable to detect formation of RecN–dsDNA complexes in EMSA using dsDNA segments up to 60-bp long (25). Alkali-lysed 2958 bp plasmid DNA was linearized with EcoRI to render molecules that were 950 nm in length as determined by AFM (Figure 2A). RecN was occasionally seen bound at random sites along the duplex DNA molecules. The linearized DNA was subjected to limited exonuclease ( Exo) digestion to obtain dsDNA molecules with 30-ssDNA extensions of about 150–450 nt single-stranded regions at both termini (Figure 2B). RecN was able to bind these ssDNA extensions (Figure 2C, left panel). To gain information on the volume of RecN–DNA complexes, the diameter (d ¼ width) of the RecN particles bound to the DNA were measured and plotted against the width of the DNA molecule to which they were bound. Linear regression adjustment of the data produced a line with an identical slope if dimeric species (d ¼ 5.7 nm calculated diameter) were bound to the DNA (d ¼ 2 nm)(27) (Figure 2C, right panel). RecN molecules were also occasionally seen bound at random sites along the DNA molecules. It seemed possible that RecN, therefore, also binds, albeit with low eﬃciency to dsDNA or, more likely, the DNA molecules puriﬁed by the alkali-lyses method contained internal single-strand nicks that were enlarged by the Exo treatment. To address this, complexes formed with these DNA preparations and a genuine ssDNA-speciﬁc binding protein, SsbA, were also visualized by AFM. SsbA bound primarily to the Exo-generated 30-ssDNA extensions but also infrequently to random sites along the linear