# DisA Limits RecG Activities at Stalled or Reversed Replication Forks
## 3 Results
### DisA Limits RecG-Mediated Fork Reversal  
Previously, it has been shown that: (i) DisA and RecG bind with significantly high affinity branched structures (e.g., HJ DNA) [21; 45]; and (ii) RecG\({}_{Eo}\) binds to stalled forks and preferentially reverses the one with a leading-strand gap [10; 11; 30; 34]. To test whether RecG shows a preference for a stalled fork with a leading-strand gap, and if DisA affects RecG-mediated fork remodeling, synthetic replicated forks with a 15-nt ssDNA gap on the parental leading-strand (forked-Lead) or on the parental lagging-strand gap (forked-Lag) were constructed. As revealed in Figure 3A,C lanes 1, 2 and 13, the indicated forked-Lead or forked-Lag substrates, intermediates or products were run to infer their mobility. The substrates have heterologous arms to prevent spontaneous branch migration. Due to that, the displaced nascent strands cannot remind to duplex DNA. Nevertheless, we can visualize the displacement of the nascent strands and the accumulation of flayed DNA (cartoons in Figure 3A,C). Figure 2: RecG unwinds HJs and nascent DNA strands from partial and complete replication fork substrates. The different radiolabeled DNA substrates (HJ-J4, Y fork, 5 '-fork, 3 '-fork or flayed DNA) were incubated with increasing RecG (15 and 30 nM) concentrations in buffer C containing 5 mM ATP (15 min at 37 \({}^{\circ}\)C). After deproteinization, substrates and products were separated by 6% PAGE in TAE buffer and analyzed by auto-radiography. The radiolabeled strands are indicated with an asterisk. In lanes 16 and 17 the [\(\gamma^{32}\)P]-labelled oligonucleotides are loaded as controls. A representative gel and below the mean \(\pm\) SD of 3 independent experiments is shown. In the presence of ATP (5 mM), DisA did not reverse a forked-Lag or forked-Lead DNA substrate (Figure 3A,C, lanes 3-5), whereas RecG (15 nM) reversed both in the absence of any accessory protein, yielding a flayed product with complete unwinding of the nascent strands from the template (Figure 3A,C, lane 6). RecG exhibited a very slight preference for the unwinding of the forked-Lead over the forked-Lag substrate (Figure 3A,C, lane 6). The RecG\({}_{\text{Eo}}\) translocase, however, shows a clear preference to reverse a forked-Lead DNA substrate [10; 30; 34]. When a fixed RecG concentration was pre-incubated with the forked-Lead or forked-Lag substrate, and then ATP and increasing DisA concentrations were added, RecG-mediated fork reversal was only slightly affected even at high DisA:RecG (3.2:1) ratios (\(\sim\)1.5-fold, \(p\) > 0.1) (Figure 3A,C, lanes 7-9). Conversely, when increasing DisA concentrations were pre-incubated with the forked-Lead or forked-Lag substrate, and then RecG and ATP were added, RecG-mediated fork reversal was strongly inhibited (\(p\) < 0.01) (Figure 3A,C, lanes 10-12). At about stoichiometric concentrations (1:1.2 DisA:RecG ratios),  
Figure 3: DisA inhibits RecG-mediated fork reversal and HJ regression. (**A,C,E**) [\(\gamma^{\text{32}}\)P] forked-Lead (**A**), [\(\gamma^{\text{32}}\)P] forked-Lag DNA (**C**), or [\(\gamma^{\text{32}}\)P] HJ-J4 DNA (0.1 nM) (**E**) was pre-incubated with increasing DisA concentrations (doubling from 12–48 nM) or a fixed amount of RecG (15 nM) in buffer B containing 10 mM MgCl\({}_{2}\) (5 min at 37 \({}^{\circ}\)C). Then, the second protein (variable DisA [RecG \(\rightarrow\) DisA], a constant amount of RecG [DisA \(\rightarrow\) RecG] or no protein) and 5 mM ATP were added and the reaction was further incubated for 15 min at 37 \({}^{\circ}\)C. The reaction was stopped, deproteinized and separated by 6% native PAGE. Gels were dried and visualized by phosphor imaging. -, no protein added; B, boiled product. (**B,D,F**) The relative amount of flayed DNA product in three independent experiments such as those shown in A, C and E was quantified, and the mean \(\pm\) SD are represented. The radiolabeled strand is indicated with an asterisk. RecG-mediated fork reversal was inhibited by \(\sim\)4-fold (Figure 3B,D).