# DisA Limits RecG Activities at Stalled or Reversed Replication Forks
## 3 Results
### DisA Inhibits the DNA-Dependent ATPase Activity of RecG  
To understand how DisA may regulate RecG helicase activity, kinetic studies of RecG-mediated ATP hydrolysis in the presence of DisA were conducted via an ATP/NADH-coupled spectrophotometric enzymatic assay. In the absence of DNA, the ATPase activity of RecG (15 nM) was undetectable, with a catalytic constant (K\({}_{\text{cat}}\)) of \(<\)0.1 min\({}^{-1}\) (Figure 4A, light blue line, Table 1). In the presence of limiting protein concentrations (5 nM, 1 monomer of RecG/ 2000-nt), RecG hydrolyzed ATP with higher efficiency upon binding to circular ssDNA (cssDNA) than to HJ DNA (Figure 4A, blue and green lines). Under the conditions used (10 mM MgCl\({}_{2}\)), the cssDNA is folded onto secondary structures that mimic flayed structures. Similar results were reported for RecG\({}_{E\alpha}\)[58; 51; 33]. The saturation of the maximal rate of ATP hydrolysis by RecG was clearly observed in our assays due to the strong ATPase activity of RecG and the consumption of some reactants. The maximal rate of ATP hydrolysis showed a continuous increase by doubling the concentration of the reactants (Figure 4A, insert, dark blue line). Figure 4: DisA inhibits the ATPase activity of RecG. (**A**) RecG (5 nM) was incubated with or without 3199-nt cssDNA or HJ-3 DNA (10 m in nt) in buffer D containing 5 mM ATP and the ATP regeneration system, and the ATPase activity was measured for 30 min. Insert, RecG (15 nM) was incubated with cssDNA in buffer D containing 5 mM ATP and 2x the ATP regeneration system, and the ATPase activity was measured for 30 min. (**B**) HJ-J3 DNA was incubated with RecG (15 nM), DisA (24-36 nM) or both in buffer D containing 5 mM ATP and the ATP regeneration system and the ATPase activity measured for 30 min. (**C**) cssDNA was incubated with RecG (15 nM), DisA (6-36 nM) or both proteins and the ATPase activity was measured. (**D**) cssDNA was pre-incubated with RecG (15 nM) or DisA (24 nM) (5 min at 37 \({}^{\circ}\)C), then DisA (RecG + ssDNA \(\rightarrow\) DisA) or RecG (DisA + ssDNA \(\rightarrow\) RecG) was added and the ATPase activity was measured. (**E** to **H**) cssDNA (**E**,**G**) or HJ-3 DNA (**F**,**H**) was incubated with RecG (15 nM) and DisA \(\Delta\)C290 (6-36 nM) (**E**,**F**) or with RecG (15 nM) and DisA D77N (6-36 nM) (**G**,**H**) and the ATPase activity was measured. All reactions were repeated three or more times with similar results, and a representative graph is shown here. DisA (36 nM) did not show any detectable hydrolysis of ATP in the presence of HJ or cssDNA (Figure 4B,C, purple line, Table 1), because hydrolysis of the two ATP molecules to form c-di-AMP cannot be measured with this ATP/NADH-coupled assay since the DAC reaction releases PPi instead of Pi. Furthermore, in the presence of an excess of ATP (5 mM), the DAC activity of DisA was reduced [42]. When HJ DNA was simultaneously incubated with RecG and DisA, RecG-mediated ATP hydrolysis was not affected at about stoichiometric RecG:DisA concentrations (molar ratio of 1:1.6) (Figure 4B, brown vs. dark blue line, Table 1). However, ATP hydrolysis was significantly reduced at a RecG:DisA 1:2.4 ratio (\(p<0.01\)) (Figure 4B, red vs. dark blue line, Table 1). The mean ATPase activity expressed as K\({}_{\text{cat}}\) values \(\pm\) SEM, estimated from the slope of the curves, can be found in Table 1.  
With cssDNA as effector, RecG (15 nM, 1 RecG monomer/666-nt) catalyzed ATP hydrolysis at the maximum rate of \(\sim\)4000 min\({}^{-1}\) (Figure 4C, dark blue line, Table 1). When DisA was simultaneously included in the reaction, RecG-mediated ATP hydrolysis was significantly inhibited (\(\sim\)4- and \(\sim\)9-fold, \(p<0.01\)) at about stoichiometric concentrations (RecG:DisA molar ratios of 1:0.8 and 1:1.6, respectively) (Figure 4C, green and brown lines, Table 1). A RecG:DisA molar ratio of 1:2.4 strongly inhibited RecG-mediated ATP hydrolysis by \(\sim\)16-fold (\(p<0.01\)) (Figure 4C, red line, Table 1). Then, it seems that DisA regulates RecG-mediated ATP hydrolysis.