# DisA Limits RecG Activities at Stalled or Reversed Replication Forks
## 3 Results  
### RecG Does Not Compete with DisA for Binding to HJ DNA  
Previously, it has been shown that: (i) DisA preferentially binds to branched intermediates than to ssDNA or dsDNA [21]; and (ii) both RecG\({}_{Eoo}\) and RecG preferentially binds with high affinity to HJ DNA [31; 45]. To test whether DisA works in concert with RecG and if both proteins co-exist on the DNA, they were purified and DNA binding assays to HJ-J3 DNA were performed (Figure 1). The integrity of the HJ-J3 DNA substrate was confirmed by assaying its sensitivity to the HJ-resolvac RecU, as described [49]. DisA bound HJ-J3 DNA with an apparent binding constant (K\({}_{\text{Dapp}}\)) of 17 \(\pm\) 1 nM and formed large molecular mass complexes or aggregates that remained trapped in the well (Figure 1A, lanes 2-4) [21]. It is likely that different protein-DNA complexes formed a net trapped in the well. From the X-ray structure, it can be predicted that an open HJ is sandwiched between two DisA molecules, as it was proposed for RuvA [50]. Here, each RuvA-like (HhH) tetrameric domain of the dumbbell-shaped structure of the DisA octamer bound to a HJ DNA molecule, and it is well-positioned to recognize another HJ DNA [21]. RecG bound the HJ-J3 DNA with a K\({}_{\text{Dapp}}\) of 4 \(\pm\) 0.3 nM, and formed mainly a G-I complex, although a minor slow-moving and diffused complex (G-II) in the presence of saturating RecG concentrations was also observed (Figure 1A, lanes 5-7). Similarly, RecG\({}_{Eoo}\) also formed a G-II complex with HJ DNA at high protein concentrations and bound DNA with similar K\({}_{\text{Dapp}}\)[31; 45]. Figure 1: DisA may coexist with RecG on HJ DNA. **(A)** For EMSA assays [\(\gamma\)\({}^{32}\)P] HJ-J3 DNA (0.1 nM in molecules) was incubated with increasing DisA (6–25 nM, lanes 2–4), RecG (7.5–30 nM, lanes 5–7) or increasing DisA and fixed RecG (lanes 14 to 16) concentrations, or pre-incubated with a fixed RecG (lanes 8–10) or increasing DisA concentrations (lanes 11–13) and then the second protein added for 15 min in buffer B containing 5 mM ATP\(\gamma\)S at 37 \({}^{\circ}\)C. The protein-DNA complexes were separated using 6% PAGE in TAE buffer and visualized by autoradiography. The RecG-HJ (G-I and G-II) and DisA-HJ (A, retained in the well) complexes are indicated. **(B)** DNAse I footprinting assays. The [\(\gamma\)\({}^{32}\)P] HJ-J3 DNA was pre-incubated with increasing RecG (15–60 nM) or DisA concentrations (30–120 nM) or increasing DisA concentrations and a fixed RecG concentration (60 nM) in buffer C containing 5 mM ATP\(\gamma\)S for 15 min at 37 \({}^{\circ}\)C, and then DNAse I was added. Samples were separated in 15% dPAGE and autoradiographed. The region protected by DisA in the presence of RecG is marked with rectangles. C denotes a HJ DNA control without DNase I treatment. In (A,B) the assay was repeated \(>\)3 times with similar results, a representative gel is shown. (C) RecG bound to HJ DNA inhibits DisA-mediated c-di-AMP synthesis. DisA (25 nM) was incubated in buffer E containing 100 \(\upmu\)M ATP in the presence of HJ-J3 DNA (125 nM) and increasing RecG concentrations (7 to 30 nM) for 30 min at 37 \({}^{\circ}\)C. Samples were separated by TLC, dried and visualized by phosphor imaging. Spots corresponding to the ATP and c-di-AMP molecules were quantified. The position of ATP, the linear pppA-pA intermediate, c-di-dAMP and the origin are indicated. A representative gel and the mean \(\pm\) SD of \(\gamma\)3 independent experiments are shown. The radiolabeled strand is indicated with an asterisk. To address whether RecG forms a ternary complex with DisA-[y\({}^{32}\)P]-HJ-J3 DNA, DNase I footprinting assays were performed. As expected, the center of the junction in the HJ-J3 DNA is in its ssDNA form, thus it was insensitive to DNase I attack (denoted as junction in Figure 1B). RecG in the apo (data not shown) or in the ATP\(\gamma\)S bound form (RecG-ATP\(\gamma\)S) did not reveal a clear DNase I footprinting (Figure 1B, lanes 2-4).