## 2 Results
### DisA Coexists with RuvAB on HJ DNA  
To test whether DisA works in concert with RuvAB, the RuvA and RuvB proteins were purified and EMSAs were performed. The RuvB-DisA interaction could not be studied by EMSA because RuvB as RuvB\({}_{Eoo}\) fails to form a stable complex with HJ DNA, even in the presence of the non-hydrolysable ATP analogue ATP\(\gamma\)S and of 0.2% glutaraldehyde addition to fix any pre-existing RuvB-DNA complex (data not shown). Similar results were previously reported [37; 41]. RuvAB forms a slow-moving complex with HJ DNA that is trapped in the well [41]. Therefore, the existence of a hypothetical DisA-HJ DNA-RuvAB complex could not be distinguished from the DisA-HJ DNA complex using EMSA (see Figure 1B). The first step in HJ resolution by the RuvAB-RecU resolvasome is the formation of the RuvA-HJ DNA complex [41]. Thus, the formation of putative RuvA-HJ DNA-DisA complexes was analyzed by EMSA. The RuvA protein binds HJ-J3 DNA preferentially in the presence of EDTA and binding is strongly reduced in the presence of 10 mM MgCl\({}_{2}\)[49; 50; 51]; therefore, the experiments were performed in the presence of 1 mM MgCl\({}_{2}\), although this is not the optimal condition for DisA binding (see Supplementary Materials and Figure S1A,B). RuvA bound [Y32P]-HJ-J3 DNA with a K\({}_{\text{Dapp}}\) of 10 \(\pm\) 2 nM, and saturating RuvA concentrations led to the formation of a slow-moving R-II complex (Figure 2D, lanes 2 and 3), which corresponds to two RuvA tetramers bound to the same HJ molecule, as observed previously in the presence of EDTA [50]. DisA bound [Y32P]-HJ-J3 DNA and formed large molecular mass complexes that mainly remained trapped in the well (Figure 2D, lane 5), as the ones observed at high Mg\({}^{2+}\) concentrations (Figure 1B). DisA seemed to facilitate RuvA-HJ DNAcomplex formation (Figure 2D, lane 6 vs. 2), and a new RuvA-HJ-J3 DNA-DisA complex (R+A) was observed (Figure 2D, lanes 7 and 9). It is likely that DisA and RuvA bound HJ DNA with a certain degree of cooperativity, and that both proteins may co-exist in the same HJ DNA molecule. To address whether DisA and RuvAB co-localize and interact with the HJ, a DNase I footprint assay was performed. The protein-[-Y32P]-HJ-J3 DNA complexes were pre-formed (5 min at 37 \({}^{\circ}\)C), and then fixed DNase I, 5 mM ATP\(\gamma\)S and MgCl\({}_{2}\) up to 10 mM were added, because DNase I requires 10 mM MgCl\({}_{2}\) for its catalytic activity (Figure 2E). The junction or cross-over region of the HJ DNA is single stranded, and thus insensitive to DNase I attack (Figure 2E, _junction_). RuvB-ATP\(\gamma\)S did not protect the HJ DNA from DNase I attack, confirming that RuvB needs to be recruited on the HJ DNA (Figure 2E, lane 2). RuvA, bound at the junction, protected both arms of the labeled HJ strand, with a footprint of \(\sim\)26-nt, and revealed some hypersensitive sites (Figure 2E, lanes 3 and 4, frame and asterisks). RuvA even protected the DNA substrate from the observed spontaneous cleavage (see lane C, control). In the presence of stoichiometric RuvB-ATP\(\gamma\)S and RuvA, HJ-J3 DNA was protected from DNase I attack, with an extended footprint of \(\sim\)40-nt (Figure 2E, framed lanes 5 and 6). This extended footprint was also observed for the RuvAB\({}_{Eco}\) complex [52]. DisA binding showed an extended region of protection from DNase I attack (40-45 bp DNA) (Figure 2E, lanes 7 and 8) [27]. This is consistent with the hypothesis that the DisA HhH domains interact with the different arms of the HJ, forming large complexes as observed above. These results indirectly suggested that DisA could prevent degradation of the extruded nascent strands of a reversed fork. At present, we cannot rule out that more than one DisA octamer is recruited to the HJ DNA. When DisA was added to pre-formed RuvAB-HJ DNA complexes or vice versa, at a DisA:RuvAB molar ratio of 1.5:1 to 3:1, an extended region of protection from DNase I attack, similar to those for DisA, was observed (Figure 2E, lanes 5-8 vs. 9-16).