## 2 Results
### DisA Interacts with RuvB  
A genetic interaction of DisA with the branch migration translocases (RuvAB or RecG) in response to DNA damage was inferred from survival assays [13]. The RecG enzyme fails to form stable complexes with DisA [27]. Here, we analyzed if RuvAB interacts with DisA. To test this, a bacterial two-hybrid system was used (see the material and methods) (Figure 2A-C).  
Figure 2: DisA interacts with RuvB and may contribute to fork protection. (**A–C**) Bacterial two-hybrid interaction assays were done, co-transforming the pair of plasmids expressing full-length DisA, DisA \(\Delta\)C290, RuvB, or RuvA fused at the N- or C-terminus, to either the T18 or T25 domain of the _Bordetella_ adenylate cyclase. A positive interaction was observed by the appearance of blue color. Experiments were repeated at least 3 times, and representative photographs are shown. In the plates, four serial dilutions of transformed cells were spotted. (**D**) DisA binds RuvA-HJ DNA complexes. [Y=32P] HJ DNA was incubated with the indicated RuvA and/or DisA concentrations for 15 min in buffer C containing 1 mM MgCl\({}_{2}\) at 37 \({}^{\circ}\)C. The protein-HJ DNA complexes were separated by 6% native  
PAGE. -, no protein added. RuvA bound to HJ forms two types of complex (R-I and R-II), DisA-HJ mainly one type of complex (A) and RuvA-HJ-DisA (R+A) complexes. (**E**) DNase I footprint analyses. [Y-32P] HJ DNA was pre-incubated with a fixed amount of RuvB (80 nM), and increasing concentrations of RuvA (40 and 80 nM) or DisA (60 and 120 nM) in buffer C containing 5 mM ATP\(\gamma\)S and 10 mM MgCl\({}_{2}\) (for 15 min at 37 \({}^{\circ}\)C). Then, the second protein (DisA or RuvAB) was added, and the reaction incubated (15 min at 37 \({}^{\circ}\)C). Finally, DNAse I was added. C, the HJ DNA control without DNase I, and in lanes 1 and 17 with DNase I treatment. The regions protected by the individual proteins or by both proteins are marked with rectangles. The order of protein addition is indicated at the top. The position of the ssDNA crossover is indicated as _'junction'_. The hypersensitive sites characteristic of RuvA binding are highlighted by an asterisk. Experiments were repeated at least 3 times, and a representative gel is shown. (**F**) PFGE of _wt_ and _AldisA_ cells after treatment or not with 10 mM MMS for 20 min before the preparation of plugs. *, DSB compression zone (above 600 kb), smaller DSBs are detected as a smear. Markers used are Lambda PFG ladder (M1) and Lambda DNA _Hind_III digest (M2). (**G**) Quantification of the chromosomal fragmentation. The increase in DSBs was plotted relative to the _wt_ untreated condition, which is given a value of 1. Results are the mean plus the SD of three independent experiments.  
The coding sequence of the genes was fused to the 5\({}^{\prime}\) or 3\({}^{\prime}\) sequence of either the T18 or T25 region of the _Bordetella_ adenylate cyclase gene, as described [18]. Using this system, we confirmed that DisA, RuvA, or RuvB interact with themselves (Figure 2A-C), because such interaction activates the cAMP-bound catabolite activator protein to induce the expression of \(\beta\)-galactosidase. This leads to the appearance of blue-colored colonies, due to the breakdown of X-gal in the medium, as observed for the Zip control (Figure 2A-C). We observed that the interaction of DisA with RuvB induces \(\beta\)-galactosidase expression (Figure 2A), whereas a physical interaction of DisA with RuvA was not deduced (Figure 2C).  
The DisA HhH domains structurally resemble that of RuvA [22]. To evaluate whether DisA interacts with RuvB through its C-terminal HhH RuvA-like DNA-binding domain, the _disA_AC290 mutant gene [18] was fused to the T18 or T25 regions. The interaction of RuvB with DisA AC290 variant induced \(\beta\)-galactosidase expression to levels comparable to that of the Zip control (Figure 2B). It is likely that the DNA-binding domain of DisA is dispensable for its interaction with RuvB.