## 2 Results  
### DisA Preferentially Birds DNA at High Mg\({}^{2+}\) Concentrations  
Single-molecule studies revealed that: (i) the dynamic movement of DisA pauses at a RecA-bound lesion-containing gap in sporulating cells [18]; (ii) DisA becomes static in the \(\Delta\)_recU_ context, where branched intermediates accumulate [23]; and (iii) DisA-mediated c-di-AMP synthesis is unaffected by duplex DNA but is inhibited by branched intermediates [22]. Thus, it is likely that octameric DisA, which consists of two peripheral quartets of helix-hairpin helix (HhH) domains connected to the central DAC domains by a helical spine [22], preferentially binds to stalled or reversed forks in a universe of duplex chromosomal DNA through its HhH domains that resemble that of RuvA. To characterize the specific DNA structure recognized by DisA, DNA binding was analyzed by electrophoretic mobility shift assays (EMSAs). The addition of 0.2% glutaraldehyde prior to separating the DisA-DNA complexes, however, was necessary to detect and visualize them. In the presence of physiological Mg\({}^{2+}\) concentrations, DisA bound both dsDNA and HJ-J3 DNA with similar apparent DNA binding constant (K\({}_{\text{Dapp}}\)) values (2.6 \(\pm\) 0.3 nM and 3.0 \(\pm\) 0.5 nM, respectively), but the complexes formed with these two substrates were different (Figure 1A,B). A fast-moving complex was observed with dsDNA, whereas DisA-HJ-J3 complexes tend to remain trapped in the well even at low acrylamide concentrations, suggesting that the RuvA-like HhH tetrameric domains arranged at each side of the dumbbell-shaped DisA octameric structure interact differently with duplex and HJ-J3 DNA [22]. DisA also bound flayed DNA (unreplicated fork DNA) and single-stranded (ss) DNA with a similar high affinity (K\({}_{\text{Dapp}}\) 3.2 \(\pm\) 0.2 nM and 3.3 \(\pm\) 0.1 nM, respectively) (Supplementary Materials Figure S1A), and formed complexes that remained trapped in the well (data not shown). This unexpected result suggests that (an) auxiliary protein(s) or cofactor specifically recruits DisA to the stalled fork, or HJ DNA. Alternatively, we did not use the proper conditions for DisA. To test the latter hypothesis, we searched for the optimal conditions for the DAC activity of DisA. DisA-mediated c-di-AMP synthesis was not detected (_p_ < 0.01) in the absence of Mg\({}^{2+}\) ions (presence of 5 mM EDTA). In the presence of 1 mM MgCl\({}_{2}\), DisA poorly catalyzed c-di-AMP synthesis, whereas the DAC activity of DisA was significantly increased in the presence of 10 mM MgCl\({}_{2}\) (Figure S2A). In the presence of 10 mM MgCl\({}_{2}\), DisA-mediated c-di-AMP synthesis was inhibited by the addition of HJ-J3 DNA (_p_ < 0.01) but not by dsDNA (_p_ > 0.1) (Figure S2B) [22]. Similarly, binding to DNA was best at high than at low MgCl\({}_{2}\) concentrations or in the presence of EDTA (_Annex 1_, Figure S1A,B). It is likely that in the presence of physiological Mg\({}^{2+}\) concentrations (10 mM), DisA forms a stable macro-complex with HJ-J3 DNA that is not entering in a gel and suppresses its DAC  
Figure 1: DisA binding to DNA. [\(\gamma\)-32P] dsDNA (**A**) or [\(\gamma\)-32P] HJ DNA (**B**) was incubated with increasing DisA concentrations (doubling from 0.45 to 60 nM) in buffer C containing 10 mM MgCl\({}_{2}\), and after fixation, complexes were detected by EMSA. Experiments were repeated at least 3 times. A representative gel and below the mean % of DNA bound and its SD are shown. A-I denotes complexes entering the gel and A-II denotes complexes retained in the well. (**C**) ATP or c-di-AMP barely affects DisA binding to HJ DNA. [\(\gamma\)-32P] HJ DNA was incubated with increasing DisA concentrations (15 min at 37 \({}^{\circ}\)C) in buffer C containing 10 mM MgCl\({}_{2}\) or 10 mM MgCl\({}_{2}\) and 500 \(\mu\)M ATP or c-di-AMP (15 min at 37 \({}^{\circ}\)C). The complexes, detected by EMSA, were quantified. Results are shown as the mean \(\pm\) SEM of \(>\)3 independent experiments. activity.