## 2 Results
### DisA Camnot Activate RuvAB to Catalyze Fork Reversal  
In _E. coli_, HJ intermediates accumulate in \(\Delta\)_ruvAB_ cells but not in the \(\Delta\)_ruvAB_\(\Delta\)_recG_ context [54], suggesting that RuvAB\({}_{E\alpha}\) by itself may not reverse a stalled fork. To test whether RuvAB converts a stalled fork with a gap in the leading or in the lagging strand onto a reversed fork, and if DisA affects the rate of such conversion, two synthetic stalled replication forks, with a 15-nt ssDNA gap either on the leading strand (forked-Lead) or on the lagging strand (forked-Lag), were used. These substrates have heterologous arms to prevent spontaneous branch migration; thus, if RuvAB mediates fork reversal, the resulting product should migrate as a flayed DNA (unreplicated fork) (Figure 3).  
Figure 3: DisA action on RuvAB translocace activity. (**A,B**) RuvAB fails to convert a gapped stalled fork into a reversed one. [\(\gamma\)-\({}^{32}\)P] forked-Lead (**A**) or [\(\gamma\)-\({}^{32}\)P] forked-Lag DNA (**B**) was pre-incubated with increasing DisA concentrations (doubling from 12–48 nM) or a fixed amount of RuvAB (15 nM) in buffer C containing 10 mM MgCl\({}_{2}\) (15 min at 37 \({}^{\circ}\)C). Then, the second protein (variable DisA [RuvAB \(\rightarrow\) DisA] or a constant amount of RuvAB [DisA \(\rightarrow\) RuvAB]) and 5 mM ATP were added, and the reaction was further incubated (15 min at 37 \({}^{\circ}\)C). The reaction was stopped, deproteinized, and separated by 6% native PAGE. -, no protein added; B, boiled forked substrate (lane 1); *, radiolabelled strand; C, expected products of the helicase activity loaded as running position controls. Representative gels are shown. (**C**) DisA inhibits RuvAB-mediated fork restoration. [\(\gamma\)-\({}^{32}\)P] HJ-1A DNA was pre-incubated with increasing DisA concentrations or a fixed amount of RuvAB in buffer C containing 10 mM MgCl\({}_{2}\) (15 min at 37 \({}^{\circ}\)C). Then, the second protein (variable DisA [RuvAB \(\rightarrow\) DisA] or a constant amount of RuvAB [DisA \(\rightarrow\) RuvAB]) and 5 mM ATP were added, and the reaction was further incubated (15 min at 37 \({}^{\circ}\)C).  
The reaction was stopped, deproteinized, and separated by 6% native PAGE. -, no protein added; B, boiled substrate. The percentage of remaining substrate (H)) is indicated at the bottom. (**D**) The relative amount of flayed DNA produced in three independent experiments as the one shown in (**C**) was quantified. Values are represented as the mean \(\pm\) SEM.  
In the presence of ATP, RuvAB (15 nM) did not unwind forked-Lead or forked-Lag DNA, and a flayed intermediate that would be the product of the backwards pushing of the replicated fork to displace both nascent strands was not observed (Figure 3A,B, lane 6). In contrast, when the branch migrating translocase RecG (15 nM) was incubated with these forked-Lead or forked-Lag DNA substrates and ATP, the flayed DNA product was observed [27].  
To test whether DisA changes this outcome by its interaction with RuvB, increasing DisA concentrations were added to the reactions with the forked-Lead or forked-Lag DNA substrates. As expected, DisA does not show any activity over these substrates (Figure 3A,B, lanes 3-5). Independently of the order of protein addition, DisA did not facilitate RuvAB-mediated formation of reversed forks by branch migrating these DNA structures (Figure 3A,B, lanes 7-12 vs. 6). Similarly, stalled forks are poor substrates for RuvAB\({}_{E\alpha}\) in vitro [31].