## 2 Results
### DisA Suppresses the DNA-Dependent ATPase Activity of RuvAB  
To understand the molecular basis of the inhibition of the RuvAB motor, we tested whether DisA inhibits RuvAB-mediated hydrolysis of ATP. As previously reported for RuvA\({}_{E\alpha}\) or RuvA [37; 41; 55], RuvA lacked any ATPase activity (\(\mathrm{K_{cat}}\) of \(<\)0.1 min\({}^{-1}\)) in the presence or absence of ssDNA (Table 1). In the absence of DNA, RuvB or the RuvAB complex (15 nM) hydrolyzed ATP with a similar \(\mathrm{K_{cat}}\) (\(\sim\)280 \(\pm\) 7 min\({}^{-1}\)) (Table 1). Similarly, RuvB\({}_{E\alpha}\) can hydrolyze ATP in the absence of any DNA [37; 55]. The DAC activity of DisA does not form ADP, the product that our assay is measuring [18].  
In the presence of circular pGEM-3 Zf(+) ssDNA (cssDNA, 10 \(\upmu\)M in nt), the ATPase activity of RuvAB (1 RuvAB complex/666-nt) was stimulated by \(\sim\)5-fold (\(p<0.01\)), but it only increased \(\sim\)2-fold (\(p<0.05\)) in the presence of HJ-J3 DNA (10 \(\upmu\)M in nt) (Figure 4A vs. Figure 4B, dark blue line, Table 1), as observed for RuvAB\({}_{Eco}\)[31]. In the presence of 10 mM Mg\({}^{2+}\), cssDNA adopts secondary structures with single- (mimicking an unreplicated fork) and double-hairpin motifs (analogous to a HJ). It is likely that these structures mimic the preferred structures for RuvAB binding (Figure 4A vs. Figure 4B), as earlier reported for RuvAB\({}_{Eco}\)[56].  
In the presence of cssDNA and limiting DisA concentrations (6 nM, \(\sim\)2 DisAs/cssDNA molecule), RuvAB-mediated ATP hydrolysis was significantly reduced (by \(\sim\)4-fold, \(p<0.01\)) (Figure 4A, orange line, Table 1). Moreover, in the presence of nearly stoichiometric DisA concentrations (12 nM) with respect to RuvAB, the maximal rate of ATP hydrolysis was comparable to that of RuvAB in the absence of DNA (\(p>0.1\)) (Figure 4A, green vs. dark blue line, Table 1). Similarly, in the presence of HJ-J3 DNA and DisA, the rate of RuvAB-mediated ATP hydrolysis was comparable to that of RuvAB in the absence of DNA (\(p>0.1\)) (Figure 4B, brown vs. dark blue line, Table 1). It is likely that DisA interacts with and inhibits the ssDNA-dependent activity of RuvB (Figure 2A,B), but it does not affect the DNA-independent ATPase activity of RuvB (or of the RuvAB complex).  
\begin{table}
\begin{tabular}{c c} \hline \hline
**Protein \(\pm\) DNA** & **K\({}_{\text{cat (min${}^{-1}$)}}\)a** \\ \hline RuvA (15 nM), no DNA & \(<\)0.1 \\ RuvB (15 nM), no DNA & \(283\pm 5\) \\ RuvAB (15 nM), no DNA & \(280\pm 7\) \\ RuvAg + HJ DNA & \(660\pm 13\) \\ RuvAB + HJ DNA + 6 nM DisA b & \(645\pm 11\) \\ RuvAB + HJ DNA + 12 nM DisA c & \(554\pm 9\) \\ RuvAB + HJ DNA + 24 nM DisA d & \(321\pm 11\) \\ RuvAB + HJ DNA + 6 nM DisA D77N b & \(651\pm 11\) \\ RuvAB + HJ DNA + 12 nM DisA D77N c & \(578\pm 7\) \\ RuvAB + HJ DNA + 24 nM DisA D77N d & \(354\pm 9\) \\ RuvAB + HJ DNA + 6 nM DisA \(\Delta\)C290 b & \(640\pm 15\) \\ RuvAB + HJ DNA + 12 nM DisA \(\Delta\)C290 c & \(547\pm 11\) \\ RuvAB + HJ DNA + 24 nM DisA \(\Delta\)C290 d & \(351\pm 10\) \\ \hline RuvAB + ssDNA & \(1253\pm 19\) \\ RuvAB + ssDNA + 6 nM DisA b & \(405\pm 8\) \\ RuvAB + ssDNA + 12 nM DisA c & \(329\pm 4\) \\ RuvAB + ssDNA + 24 nM DisA d & \(301\pm 5\) \\ RuvAB + ssDNA + 6 nM DisA D77N b & \(433\pm 13\) \\ RuvAB + ssDNA + 12 nM DisA D77N c & \(350\pm 9\) \\ RuvAB + ssDNA + 24 nM DisA D77N d & \(330\pm 8\) \\ RuvAB + ssDNA + 6 nM DisA \(\Delta\)C290 b & \(461\pm 14\) \\ RuvAB + ssDNA + 12 nM DisA \(\Delta\)C290 c & \(375\pm 11\) \\ RuvAB + ssDNA + 24 nM DisA \(\Delta\)C290 d & \(341\pm 9\) \\ \hline \hline \end{tabular}
\end{table}
Table 1: RuvAB rates of ATP hydrolysis.