## 2 Results
### RuvAB- or RecU-Bound HJ-J3 DNA Blocks DisA-Mediated C-Di-AMP Synthesis  
DisA catalyzes the synthesis of the essential c-di-AMP second messenger that regulates a wide variety of physiological functions and plays a central role in virulence in bacteria of the Firmicutes phylum [16]. DisA inhibits RuvAB- and RecU-mediated HJ processing (Figures 3D and 5D), and in the presence of HJ DNA, DisA-mediated c-di-AMP synthesis is inhibited [22; 23]. We tested whether RuvAB or RecU regulates the DisA DAC activity at limiting ATP concentrations (K\({}_{\text{m}}\) 151 \(\pm\) 1.4 uM) [27] to reduce the branch migrating activity of RuvAB that would disassemble the HJ DNA. Under the conditions used to detect radiolabeled [\(\alpha\)-32P]-c-di-AMP, [\(\alpha\)-32P]-ADP is poorly separated from the [\(\alpha\)-32P]-ATP substrate; therefore, RuvAB-mediated ATP hydrolysis cannot be detected in our assay. In the presence of 100 uM ATP and 10 mM MgCl\({}_{\text{2}}\), DisA converted 70-80% of the ATP substrate into product (c-di-AMP), and traces of the pppA-pA intermediate were detected (Figure 6A,B, lane 2), as described [22; 23]. The addition of RuvAB or RecU (30 nM) marginally affected the DAC activity of DisA (\(p\) > 0.1) (Figure 6A,B, lane 7 vs. 2). As expected [22; 23], in the presence of a fixed HJ-J3 DNA concentration, DisA-mediated c-di-AMP synthesis was reduced by \(\sim\)3-fold (\(p\) < 0.05) (Figure 6A,B, lanes 3 vs. 2). In the presence of HJ-J3 DNA and RuvAB or RecU, several outcomes can be expected when  
Figure 6: HJ DNA and RuvAB or RecU inhibits DisA-mediated c-di-AMP synthesis. (**A**,**B**) A fixed DisA concentration was incubated alone (lane 2), with HJ DNA (125 nM) (lane 3), increasing RuvAB (**A**) or RecU (**B**) concentrations (7, 15, or 30 nM) and HJ DNA (lanes 4–6), or with RuvAB (**A**) or RecU (**B**) (30 nM, lane 7) in buffer D containing 100 uM ATP (at a ratio of 1:2000 [\(\alpha\)32P]-ATP:ATP) and 10 mM MgCl\({}_{\text{2}}\) (30 min at 37 \({}^{\circ}\)C). In lane 8, DisA was pre-incubated with HJ DNA (5 min at 37 \({}^{\circ}\)C) and then a fixed concentration of RuvAB (**A**) or RecU (**B**) and ATP were added, and the reaction incubated (30 min at 37 \({}^{\circ}\)C). In lane 9, RuvAB or RecU was pre-incubated with HJ DNA and then DisA and ATP were added, and the reaction was incubated (30 min at 37 \({}^{\circ}\)C). In lane 10, a control reaction with only RuvAB or RecU (30 nM) is shown. The substrates and products were separated by TLC, and the spots quantified. The position of ATP, linear pppA, c-di-AMP, and the origin are indicated. At least three independent experiments were performed, a representative plate, and the mean % of c-di-AMP produced and its SD are shown. the DisA DAC activity is assayed. First, RuvAB (or RecU) may displace DisA by competing for binding or by unwinding or cleaving, respectively, the HJ DNA, leading to the recovery of the DAC activity of DisA. Second, RuvAB (or RecU) may stabilize or relocate DisA on the HJ-J3 structure, thus additively affecting DisA-mediated c-di-AMP synthesis. Third, DisA bound to HJ DNA may not interact with RuvB, and the DAC activity is inhibited as in the presence of only HJ DNA. We found that in the presence of HJ-J3 DNA and a limiting RuvAB concentration (RuvAB:DisA 0.3:1 molar ratio), the DAC activity of DisA was additively inhibited (\(\sim\)6-fold) (\(p<0.01\)), suggesting that RuvAB can stabilize or relocate DisA on HJ-J3 DNA (see above). At RuvAB:DisA ratios approaching stoichiometry (0.6:1 and 1.2:1), RuvAB slightly reversed this negative effect, but the DisA DAC activity was still inhibited when compared with HJ DNA alone (Figure 6A, lane 3 vs. 5 and 6). To understand this mechanism of inhibition, the order of protein addition was varied. When DisA was pre-incubated with HJ-J3 DNA, and then RuvAB at a 1.2:1 RuvAB:DisA molar ratio was added, DisA-mediated c-di-AMP synthesis was strongly inhibited, suggesting that RuvAB stabilizes or relocates DisA on the HJ-J3 structure and this further impedes the DAC activity.