## 4 Materials and Methods
### DNA Binding, HJ Branch Migration, and Cleavage Assays  
DNA binding was assayed by EMSA using different [\(\gamma\)32P]-labelled DNA substrates (0.2 nM in molecules). The radiolabeled strand is indicated with an asterisk. The binding was performed in buffer C (50 mM Tris-HCl pH 7.5, 50 mM NaCl, 1 mM DTT, 0.05 mg/mL BSA, 5% glycerol) containing 1 or 10 mM MgCl\({}_{2}\) or 5 mM EDTA and when indicated a nucleotide cofactor. Reactions were incubated for 15 min at 37 \({}^{\circ}\)C. Prior to loading, 0.2% glutaraldehyde was added to stabilize the complexes. Protein-DNA complexes were separated using 6% PAGE in TAE buffer and visualized by autoradiography. Autoradiography films were scanned, and the ImageJ software (NIH, Bethesda, MD, USA) was used to determine the signal from each band and obtain the apparent binding constant (K\({}_{\text{Dapp}}\)) values at the protein concentration that gives 50% of DNA-protein complexes. _t_-tests were applied to analyze the statistical significance of the data. The reaction conditions for DNase I footprint experiments were the same as for EMSA. DNase I treatment was performed as described [62]. The samples were resuspended in loading buffer [80% (\(v/v\)) formamide, 10 mM NaOH, 1 mM EDTA, 0.1 % (\(v/v\)) bromophenol blue, and 0.1% (\(v/v\)) xylene cyanol], separated in 15% denaturing PAGE (dPAGE), and autoradiographed. For the size control marker, ladders obtained with the chemical sequencing reaction (G + A) on the same DNA fragments were used. Cleavage of HJ-J DNA (labeled on arm 1) at the indicated concentrations of RecU was assayed for 30 min at 37 \({}^{\circ}\)C in buffer C containing 10 mM MgCl\({}_{2}\). When indicated, increasing concentrations of DisA were added. After deproteinization by the addition of one-fifth volume of stop mix (5% SDS, 100 mM EDTA, 5 mg/mL proteinase K) and further incubation for 10 min at 37 \({}^{\circ}\)C, the products of the cleavage were analyzed by 15% denaturing PAGE and autoradiography. _t_-tests were applied to analyze the statistical significance of the data. In a standard branch migration assay, [\(\gamma\)32P]-labelled HJ-J4 DNA (0.1 nM in molecules) was incubated with RuvAB in buffer C containing 10 mM MgCl\({}_{2}\) and 5 mM ATP for 30 min at 37 \({}^{\circ}\)C. When indicated, increasing concentrations of DisA were added. Reactions were terminated by adding one-fifth volume of stop mix (5% SDS, 100 mM EDTA, 5 mg/mL proteinase K) and further incubation for 10 min at 37 \({}^{\circ}\)C to deproteinize the sample. Unwound products were analyzed by 6% PAGE in TAE buffer and phosphorimaging. Signals of substrates and products were quantified with the ImageJ software (NIH). \(t\)-tests were applied to analyze the statistical significance of the data. The following are available online at [[https://www.mdpi.com/article/10.3390/ijms222111323/s1](https://www.mdpi.com/article/10.3390/ijms222111323/s1)]([https://www.mdpi.com/article/10.3390/ijms222111323/s1](https://www.mdpi.com/article/10.3390/ijms222111323/s1)), Figure S1: Affinity of DisA for different DNA substrates, Figure S2: DisA DAC activity requirements; Figure S3: Extent of spontaneous chromosomal fragmentation in the \(\Delta\)_rccU_ context. Conceptualization, S.A. and J.C.A. ; formal analysis, validation and investigation, C.G., R.T. and B.C. ; data curation, C.G., R.T., B.C., S.A. and J.C.A. ; writing--original draft preparation, S.A. and J.C.A. ; writing--review and editing, R.T., B.C., S.A. and J.C.A. ; supervision, S.A. and J.C.A. All authors have read and agreed to the published version of the manuscript. This research was funded by Ministerio de Ciencia e Innovacion/Agencia Estatal de Investigacion (MCel/AEI)/FEDER, EU). Grant No. PGC2018-097054-B-I00 to S.A and J.C.A. Not applicable. The datasets analyzed or generated during the study are avalaible upon request. We are grateful to [PERSON] and [PERSON] for the communication of unpublished results, and to [PERSON] for her contribution in the early steps of the project.