# DisA Limits RecG Activities at Stalled or Reversed Replication Forks
## 3 Results
### RecG on HJ DNA Blocks DisA-Mediated c-di-AMP Synthesis  
Previous reports have shown that: (i) in unperturbed exponentially growing cells, DisA forms a static complex on the nucleoid in \(\Delta{recG}\) cells, where significant amounts of branched intermediates accumulate, but maintains its dynamic movement in wt cells [27]; (ii) the _disA_ gene is epistatic to _recG_ in response to DNA bulky and non-bulky lesions [20; 27]; and (iii) DisA-mediated c-di-AMP synthesis is decreased upon binding to HJ DNA, whereas DisA bound to duplex DNA barely reduces the synthesis of the essential c-di-AMP, which regulates a wide variety of physiological functions [21; 22; 27]. At least two mutually exclusive conditions for the putative RecG and DisA interactions at the HJ DNA can be envisioned. First, RecG displaces DisA from the HJ-J3 DNA, with subsequent recovery of its DAC activity. Second, RecG binds to and stabilizes the HJ-J3 DNA, and relocates DisA to it, further reducing DisA-mediated c-di-AMP synthesis. To test whether RecG stabilizes a preferred conformer and re-locates DisA to it or if RecG itself affects DisA-mediated c-di-AMP synthesis, the DAC activity of DisA was analyzed.  
At 5 mM ATP, the maximal unwinding activity of RecG was observed [45]. However, steady-state kinetic analysis of DisA (25 nM) revealed that it efficiently converted two ATP molecules into c-di-AMP in the presence of 10 mM Mg\({}^{2+}\), and its DAC activity was saturated at \(\sim\)300 \(\upmu\)M ATP (Supplementary Figure S1). Thus, we have used conditions (100 \(\upmu\)M ATP) in which DisA maintains its DAC activity, but RecG-mediated unwinding of HJ DNA is poorly detected. Indeed, after 30 min incubation, RecG (30 nM) did not catalyze \(c\)-di-AMP synthesis (Figure 1C, lane 8). It is worth mentioning that under the conditions used to detect [\(\alpha^{32}\)P]-c-di-AMP, [\(\alpha^{32}\)P]-ADP is poorly separated from the [\(\alpha^{32}\)P]-ATP substrate, therefore, RecG-mediated hydrolysis of ATP cannot be observed [27]. In the presence of 100 \(\upmu\)M ATP (at a ratio of 1:2000 [\(\alpha^{32}\)P]-ATP:ATP), DisA (25 nM) efficiently synthesized c-di-AMP, with \(\sim\)75% of the radiolabeled ATP converted to c-di-AMP. Nearly equimolecular concentrations of RecG (30 nM) did not significantly affect the DAC activity of DisA (Figure 1C, lane 2 vs. 7).  
Addition of HJ-J3 DNA (125 nM), at a 5:1 stoichiometry relative to DisA, reduced c-di-AMP synthesis (\(\sim\)2-fold, \(p<\) 0.01) (Figure 1C, lane 3 vs. 2), as earlier reported [21; 27]. In the presence of HJ-J3 DNA, limiting RecG concentrations (7 nM, a RecG:DisA molar ratio of 0.3:1) significantly blocked the DAC activity of DisA by \(\sim\)11-fold (\(p<\) 0.01) (Figure 1C, lane 4). This suggests that RecG, under these experimental conditions that do not allow DNA unwinding, further inhibits DisA-mediated c-di-AMP synthesis. Higher RecG concentrations did not reverse such inhibition (Figure 1C, lanes 5-6). It is likely that RecG bound to the HJ-J3 DNA relocates DisA to a position in which DisA-mediated c-di-AMP synthesis is blocked, rather than competing or displacing DisA from the HJ DNA, displacement that would counteract the negative effect of HJ DNA on DisA-mediated c-di-AMP synthesis.  
We interpreted this observation as that RecG contributes to limit c-di-AMP synthesis, and this effect indirectly blocks cell proliferation. This is consistent with the observation that: (i) in response to MMS-induced DNA lesions, the c-di-AMP levels decreased to levels comparable to that in the absence of DisA [26]; (ii) low c-di-AMP levels indirectly increase (p)ppGpp synthesis [52; 53]; and (iii) an increase of ppPGPp levels inhibits DNA replication by inhibiting DNA primase activity [54].