## 2 Results
### DisA Does Not Interact with RecU  
With few exceptions, the RecU HJ resolvase constitutes, together with the RuvAB complex, the resolvasome in bacteria of the Firmicutes and Tenericutes phyla [41]. Genetic studies suggest that _disA_ is epistatic to the _recA_ or _recU_ gene in response to DNA damage [13; 23]. The RecA protein physically interacts with both DisA and RecU [18; 57]. Upon cell envelope stress, the expression of both the _disA_ and the _recU_ genes is significantly increased [35; 36], suggesting a temporal and functional link between them. Therefore, a functional interaction of DisA and RecU was explored. First, we analyzed whether RecU and DisA physically interact using the bacterial two-hybrid system. RecU interacts with itself, in agreement with the X-ray data that show that the protein is a dimer [42]. A stable interaction of RecU with DisA, however, was not observed (Figure 5A). Figure 5: DisA does not interact with RecU but inhibits RecU-mediated HJ cleavage. (**A**) DisA interaction with RecU was not observed by the bacterial two-hybrid interaction assay. (**B**) [\(\gamma\)-32P] HJ DNA was pre-incubated with increasing DisA concentrations (doubling from 12–48 nM) or RecU (doubling from 0.25–1 nM) for 15 min in buffer C containing 1 mM MgCl\({}_{2}\) (5 min at 37 \({}^{\circ}\)C). Then,  
the second protein (variable DisA or a constant amount of RecU) was added and the reaction was incubated (15 min at 37 \({}^{\circ}\)C). The protein-H) DNA complexes were separated by 6% native PAGE. -, no protein added. The RecU-H) (U) and DisA-H) (A) complexes are shown. The order of protein addition is indicated at the top. (**C**) Autoradiogram showing a footprint analysis of the binding of RecU and DisA to H) DNA. [\(\gamma\)-\({}^{32}\)P] HJ DNA was pre-incubated with increasing concentrations of RecU (50 and 100 nM) or DisA (60 to 120 nM) (5 min at 37 \({}^{\circ}\)C) in buffer C containing 1 mM MgCl\({}_{2}\). Then, the second protein (RecU 50 nM) was added, and the reaction incubated (15 min at 37 \({}^{\circ}\)C). After that, DNase I and MgCl\({}_{2}\) up to 10 mM were added. C, no DNase I was added. The position of the ssDNA crossover is indicated as _junction_ and RecU-mediated cleavage of the [\(\gamma\)-\({}^{32}\)P]-labelled arm is indicated by an \({}^{\star}\). The G + A marker is indicated. Experiments were repeated at least 3 times, and a representative gel is shown. (**D**) [\(\gamma\)-\({}^{32}\)P] HJ DNA labelled on arm 1 (denoted by \({}^{\star}\)) was pre-incubated with DisA (doubling from 24-96 nM) or RecU (100 nM) (5 min at 37 \({}^{\circ}\)C) in buffer C containing 1 mM MgCl\({}_{2}\). Then, the second protein (variable DisA [RecU \(\rightarrow\) DisA]) or a constant amount of RecU [DisA \(\rightarrow\) RecU]), and MgCl\({}_{2}\) up to 10 mM were added, and the reaction was incubated (15 min at 37 \({}^{\circ}\)C). The reaction was stopped, deproteinized, and analyzed by 15% denaturing PAGE, no protein added. The relative amount of cleaved DNA in three independent experiments was quantified as described, and a representative gel, the mean % of cleaved DNA, and its SD are shown. To analyze whether DisA modulates the mechanism of RecU-mediated HJ resolution, the RecU HJ resolvase was purified, and DNA binding was analyzed. In the presence of 1 mM Mg\({}^{2+}\), RecU specifically binds HJ DNA, and in the presence of 10 mM Mg\({}^{2+}\), RecU binds and cleaves the HJ structure when its cognate site is exposed [42; 46]. Therefore, to perform DNA binding studies, we used 1 mM MgCl\({}_{2}\), which it is not the optimal condition for DisA binding (see Supplementary Materials, and Figure S1). RecU binds HJ-J3 DNA with high affinity (K\({}_{\text{Dapp}}\) of 0.6 \(\pm\) 0.2 nM; Figure 5B, lanes 5-7), as described [46] and with \(\sim\)30-fold higher affinity than DisA under this experimental condition.