AFM images of Nd.
A. The left panel shows a typical field view of Nd at 1 nM. Elongated and round structures are observed. Different Nd tetramers will deposit on the mica surface at different orientations affecting their overall appearance. Some tetramers dissociate during the drying process and therefore some smaller structures are also apparent. The right panel shows a typical field view of Nd at 6 nm, with scaffolds of variable sizes and a long filament clearly visible.
B. The top left and middle panels show two close-up images of elongated Nd tetrameric structures at 1 nm. The top right panel shows the section analysis of the monomer, indicated by the arrow, and the bottom two panels show the section analyses along the two elongated tetramers.
C. Filaments of Nd obtained at 1 nm (top left and right) and 6 nm (bottom left) respectively. The inset shows a comparison of section analyses of the filament (red) and monomer (green) indicated by the arrows in the same view.
D. Typical field views and close-up images (insets) of scaffolds observed at higher concentrations (3 nm left panel, 6 nm right panel). Scale bars represent 200 nm.

SOXL cross-linking of Cd in the presence and absence of a ss 19mer oligonucleotide. Panel A shows SOXL cross-linking of Cd (18 μM) in the absence of oligonucleotide and control Cd in the absence of cross-linker, as indicated. Panel B shows control Cd (18 μM) in the absence of cross-linker and also cross-linking reactions with increasing concentrations of SOXL (0.5, 1.0 and 1.5 mM), at two different oligonucleotide concentrations (0.9 and 0.19 μM), as indicated. Internal cross-links as well as large cross-linked species are indicated as Cd and (Cd)X respectively. MW markers are indicated in kDa in lanes M. In the presence of the oligonucleotide large cross-linked Cd species are clearly visible whereas in the absence of DNA only internal cross-links can be seen, confirming the DNA-induced oligomerization activity of Cd. Panel C shows AFM images of Cd bound to pBR322. Large foci represent oligomers of bound Cd molecules. Plasmids remain in their original supercoiled forms and do not form OCCs. The concentration for Cd and pBR322 was 6 nm and 32 pm respectively. Scale bars represent 200 nm.

Cd is identified as a “DnaD-like protein motif” (PF04271). It is found in all DnaD proteins (DnaD proteins from some bacterial species are shown in the left column) and also in some proteins of unknown function (some typical examples are shown in the middle column). For example, Bacillus subtilis also has another protein YqaI of unknown function that contains the DnaD-like domain. Several phage proteins also contain the DnaD-like domain (right column). Some proteins have two tandem DnaD-like domains (bottom right) while orf4 of phage 7201 has a DnaD-like domain linked to a RepA-like domain (bottom right).

Oligomeric states of Nd and Cd.
A. MW distribution, c(M), of Nd and Cd from sedimentation velocity studies. Nd and Cd have mass distributions of 62 and 13 kDa, corresponding to a tetramer and monomer respectively.
B. Sedimentation equilibrium data for Nd and Cd. Only one loading concentration is shown for clarity. Data were obtained at 12 000, 16 000, 22 000 and 28 000 rpm. Residual plots of the fit to a single species are shown below each data set. Nd has an MW corresponding to a tetramer, whereas Cd is a monomer.
C. SDS-PAGE analysis showing cross-linking of DnaD with SOXL. Lanes 1 (9 μM DnaD or Nd) and 2 (4.5 μM DnaD or Nd) indicate control reactions in the absence of SOXL whereas lanes 3 [protein (18 μM)/SOXL (180 μM)] and 4 [protein (18 μM)/SOXL (504 μM)] indicate reactions with increasing relative ratios of SOXL to DnaD or Nd. Monomeric and dimeric species of DnaD are shown as (D)1 and (D)2 whereas monomeric, dimeric and tetrameric species of Nd as (Nd)1, (Nd)2 and (Nd)4 respectively. MW markers (kDa) are shown in lanes M.

Cd binds DNA. Gel shift assays using a 54mer ss oligonucleotide. DnaD and Cd exhibit DNA-binding activity whereas Nd does not. The complexes are too big to enter into the gel and stay in the wells. Lanes C show control reactions in the absence of protein.

A. Agarose shift assays to examine the effects of DnaD and its domains on supercoiled pBR322. DnaD forms large OCCs while Nd, Cd and Nd + Cd do not alter the electrophoretic mobility of the supercoiled plasmid through the agarose gel. Binding reactions are shown in the left half of all gels while the right half shows the same reactions but with proteinase K treatment before loading. All reactions were carried out at 4.34 nM pBR322 and increasing concentrations of proteins (11, 22, 34, 45, 90, 140 and 201 μM). Open circular and supercoiled plasmids are indicated by OC and SC respectively. Lanes C show controls in the absence of protein and lane M shows MW markers (10, 8, 6, 5, 4, 3, 2, 1.5, 1 and 0.5 kb from top to bottom). B. A model for DnaD-mediated DNA remodelling. DnaD consists of an extended Nd domain (green) and a DNA-interacting Cd domain (red). It forms a dimer and at higher concentrations filaments and scaffolds via Nd-mediated interactions. Cd binds to DNA and a conformational change (Cd* shown in yellow) reveals a second DNA-dependent oligomerization interface on this domain, which causes the accumulation of DnaD molecules on the DNA forming large foci.