a. Side view of S. aureus DNA gyrase core fusion structure (6FQV) with bound DNA (green). The GyrB segment is in purple (only the TOPRIM domain here) and the GyrA in blue. The C2 symmetry mate is rendered in gray. The inset shows the location of isoleucine 174 in red (E. coli numbering) which is inserted between base +8 and +9 (from scissile phosphate) and introduces a kink in the duplex. b. Top view of the same structure. The inset shows in light brown the catalytic tyrosine sandwiched between an arginine and a threonine and forming the triad RYT. c. Cartoon rendering of the DNA gyrase heterotetramer. GyrB is in purple and GyrA in blue. The segment in solid colors denote the part of the enzyme included in the S. aureus core fusion structure. Various domains are indicated, and the dashed line circles indicate the three interfaces that the transported DNA segment go through during strand passage. d. Schematic of DNA gyrase peptidic sequences, color coded as above. The core fusion construct used in structural studies is indicated.

Analysis of GyrA, GyrB and heterodimers preparations. a. Blue-Native PAGE of our GyrB, GyrA and wild-type heterodimer preparation. The bands constituted by GyrB monomers, GyrA dimers and heterodimers are indicated and were visualized by silver staining. The migration pattern was ascertained by comparison to a native marker and is consistent with mass photometry profiles below. b. Mass Photometry profile of the GyrA preparation. The histogram shows the counts of collisions events plotted against the scattering intensity, which is proportional to the MW and is calibrated against urease; the abscissa shows the molecular mass in kDa. The instrument fits the observed peaks to gaussian curves (continuous black lines) and the mean (MW) and deviation (σ) of the fitted curves are indicated on top of the peak, alongside the total count for the peak and the percentage of counts assigned to the peak with respect to the total number of fitted events. b. Mass Photometry profile of our wild-type heterodimer preparation, as above. Note that the lower the peak count, the higher the deviation. We detect a main peak at approximately the expected size for a heterodimer. c. Superimposition of the two profiles (GyrA dimer in blue and heterodimer in orange) showing the difference in mass between the heterodimer and the GyrA dimer. The two profiles were collected the same day, in the same buffer and with the same calibration.

Reconstitution of double-strand cleavage activity in the presence of free GyrB. Both cleavage (top) and supercoiling (bottom) assays were performed were performed with BA.A, BA.AF and BAF.A as indicated. For cleavage assays 5, 2.5 and 1.25 pmole of heterodimers (three lanes, from left to right for each heterodimer version, the triangle shows increasing dosage of heterodimers) were used. 8 pmole of GyrB were added to reconstitute the activity (top left panel). Omitting GyrB showed only background cleavage (top center panel). GyrB alone (top right panel) showed almost undetectable cleavage activity. For supercoiling assays the dose of each subunit was reduced, so as to keep the activity limiting in the assay. 4 pmole of GyrB was added to 1.25, 0.625 and 0.312 pmole of heterodimer, the triangle showing increasing dose of the heterodimer.

a. Schematic of the radiolabeling of the catalytic tyrosine. The peptide RYT is shown in black, and a cleavage reaction results in a covalent bond between the catalytic tyrosine and a radiolabeled DNA (in red with asterisk). Subsequent digestion with micrococcal nuclease digests most of the DNA and leaves a stub of radiolabeled nucleic acid covalently bound to the catalytic tyrosine. b. The resulting labelled gyrase polypeptide can be analyzed by SDS-PAGE and detected by exposure to a phosphor screen. The cleavage reactions contained 8 pmole of GyrB and 5 pmole of heterodimer. From left to right: wild-type heterodimer with Mg2+, without Mg2+, BAF.A (1), BA.AF (2) and BALLL.A. The upper band is the fusion polypeptide, the lower band is the GyrA polypeptide. The smear at the bottom is the bulk of the digested radiolabeled DNA.

Effect of GyrB dose on subunit exchange. 5 pmole of heterodimers (BA.A and BAF.A as indicated) were incubated with increasing amount of GyrB (triangle) in a cleavage assay. From the highest to the lowest dose: 16, 8, 4, 2, 1 and 0.5 pmole were used. At lower GyrB/heterodimer ratio, single-strand cleavage is predominant with BAF.A whereas BA.A still displays robust double-strand cleavage activity. Single-strand cleavage activity does go up marginally with the wild-type protein at the lower GyrB dose, suggesting missing GyrB on one side can lead to the formation of single-strand cleavage complexes. However, even at very low GyrB/heterodimer ratio, the majority of cleavage complexes are double-stranded.

Effect of DNA wrapping and DNA bending defective mutants on subunit exchange. a. 5, 2.5 and 1.25 pmole (triangle indicate increasing dose) of BA.A59 and BAF.A59 (as indicated) were incubated in a cleavage assay with 8 pmole of GyrB. b. 2.5, 1.25 and 0.625 pmole (triangle indicate increasing dose) of BA.A59 and BAF.A59 (as indicated) were incubated in a supercoiling assay in the presence of 4 pmole of GyrB c. 5, 2.5 and 1.25 pmole (triangle indicate increasing dose) of BA.A, BAFA and BAG.A were incubated in a cleavage assay in the presence of 8 pmole of GyrB. d. 2.5, 1.25 and 0.625 pmole (triangle indicate increasing dose) of BA.A59 and BAF.A59 (as indicated) were incubated in a supercoiling assay in the presence of 4 pmole of GyrB. Omitting GyrB from all these assays abolished either the supercoiling or cleavage respectively. The exception being BA.A59, which shows a small amount of double-strand cleavage in the absence of GyrB (Supp Fig. 6b)

Kinetics of subunit exchange. a. 5 pmole of either BA.A (blue) or BALLL.A (orange) were incubated with 8 pmole of GyrB in a cleavage assay for the indicated time. BALLL.A + GyrB reaches a level of cleavage comparable to BA.A (albeit much later, around 60 minutes, not shown). b. Quantification of the gels shown in a. the proportion of linear of the total amount of material in each lane is plotted against time. The proportion of linear is normalized as the fraction of the total amount of linear reached at 60 minutes, minus background cleavage (very low in that instance) observed at the 0 time point.

Proposed model for interface swapping. a. Cartoon schematic of two gyrase complexes in close proximity, viewed from the side. Only the core complex is shown. b. 90° rotation view. The gyrase “super-complex” is viewed from the top. c. The opening of each DNA-gate interface occurs by a sliding movement (black arrows), allowing reformation of a hybrid interface, in the center of the super-complex. The DNA is not represented.