DOI: 10.5061/dryad.w6m905qwn
title: Rapid, DNA-induced interface swapping by DNA gyrase

description: DNA gyrase, a ubiquitous bacterial enzyme, is a type IIA topoisomerase
 formed by heterotetramerisation of 2 GyrA subunits and 2 GyrB subunits, to
 form the active complex. GyrA is usually found as a dimer in solution,
 whereas GyrB can exist as a monomer. DNA gyrase is able to loop DNA around
 the C-terminal domains (CTDs) of GyrA and pass one DNA duplex through a
 transient double-strand break (DSB) established in another duplex. This
 results in the conversion of a positive loop into a negative one, thereby
 introducing negative supercoiling into the bacterial genome, an activity
 essential for DNA replication and transcription. The strong protein
 interface in the GyrA dimer must be broken to allow passage of the
 transported DNA segment and it is generally assumed that the interface is
 usually stable and only opens when DNA is transported, to prevent the
 introduction of deleterious DSBs in the genome. In this paper we show that
 DNA gyrase can exchange its DNA-cleaving interfaces between two active
 heterotetramers. This so-called interface “swapping” or “exchange” (IS)
 can occur within a few minutes in solution. We also show that bending of
 DNA by gyrase is essential for cleavage but not for DNA bindingper seand
 favors IS. interface swapping is also favored by DNA wrapping and an
 excess of GyrB. We suggest that proximity, promoted by GyrB
 oligomerization and binding and wrapping along a length of DNA, between
 two heterotetramers favors rapid interface exchange. This exchange does
 not require ATP, can occur in the presence of fluoroquinolones, and raises
 the possibility of non-homologous recombination solely through gyrase
 activity. The ability of gyrase to undergo interface swapping also
 explains how gyrase heterodimers, containing a single active-site
 tyrosine, can carry out double-strand passage reactions and therefore
 suggests an alternative explanation to the recently proposed “swivelling”
 mechanism for DNA gyrase (Gubaev, Weidlich, and Klostermeier 2016).
description: # Rapid, DNA-induced interface swapping by DNA gyrase
 [https://doi.org/10.5061/dryad.w6m905qwn](https://doi.org/10.5061/dryad.w6m905qwn) This repository contain most of the raw data presented in the article. Organization: For each figure and figure supplement, a corresponding zipped folder is available for download. They each contain a labelled file(s) for each subsection of the figure. The files format are .tif for gel picture, this is the output format from the imaging instrument, without any correction applied. These files can be used for quantification by densitometry. For the FPLC trace the file format is .txt containing the tab-separated values of the FPLC output. For the SPR and Mass Photometry data a .csv file is provided. Accompanying each figure and figure supplement is a pdf file showing the annotated uncropped pictures when appropriate. Please refer to the following key for access to specific data. Figure_1_figure_supplement_1.zip: zipped folder containing the following files Annotated_gel_Figure1_SUPP_FIgure1.pdf ----------------&gt;&gt; PDF files containing the uncropped, annotated image of the two gels presented in the figure. FPLC_AKTA_trace_Fig_1_SUPP_FIGURE_1_B.txt ----------------&gt;&gt; raw data for the FPLC trace presented in the B panel. The format is tab-separated values with 2 header lines. FPLC_AKTA_trace_Fig1_SUPP_FIGURE_1_C.txt ----------------&gt;&gt; raw data for the FPLC trace obtained with the BA.A heterodimer, presented in the C panel. The format is tab-separated values with 2 headers. FPLC_AKTA_TRACE_GyrA_Fig1_SUPP_FIGURE_1_C.txt ----------------&gt;&gt; raw data for the FPLC trace obtained with the GyrA dimer, presented in the C panel. The format is tab-separated values with 2 header lines. gel_expression_Fig1_SUPP_FIGURE_1_A.tif ----------------&gt;&gt; picture of the gel presented in the A panel. This is a coomassie-stained protein gel. format is TIF gel_filtration_gel_Fig1_SUPP_FIGURE_1_B.tif ----------------&gt;&gt; picture of the gel presented in the B panel. This is a coomassie-stained protein gel. format is TIF Figure_2.zip: zipped folder containing the following files Collision_events_Figure_2_C_and_D.csv ----------------&gt;&gt; collisions events detected by the REFEYN (1st generation) mass photometry instrument for the BA.A heterodimer (panel C and D). Format is comma-separated-vaues (csv) Collisions_events_figure_2_B_and_D.csv ----------------&gt;&gt; collisions events detected by the REFEYN (1st generation) mass photometry instrument for the GyrA dimer (panel B and D). The formatormat is comma-separated-vaues (csv) Figure_2_A_annotated.pdf ----------------&gt;&gt; PDF files showing the uncropped Blue-NATIVE PAGE gel in panel A with annotation Figure_2_A.tif ----------------&gt;&gt; Original image taken of the Blue-NATIVE PAGE gel in panel A in TIF format. Figure_2_figure_supplement_1.zip: zipped folder containing the following files. BA_A_RAW_Spectra_ist100_cid100.RAW ----------------&gt;&gt; Raw native mass spectrometry spectra for the BA.A heterodimer GyrA_RAW_Spectra_ist200_cid200.RAW ----------------&gt;&gt; Raw native mass spectrometry spectra for the GyrA dimer Figure_2_Figure_supplement_2.zip: Blot_PanelC.jpg----------------&gt;&gt; Western blotting of the material presented in the loading control wit an anti-GyrA CTD antibody BNPAGE_PanelA.jpg----------------&gt;&gt;BNPAGE (coomassie stained) original image of the gel presented in Panel A BNPAGE_PanelB.jpg----------------&gt;&gt;BNPAGE (coomassie stained) original image of the gel presented in Panel B BNPAGE_PanelC.jpg----------------&gt;&gt;BNPAGE (coomassie stained) original image of the gel presented in Panel C (loading cotrol) BNPAGE_PanelD_time0.jpg----------------&gt;&gt;BNPAGE (coomassie stained) original image of the gel presented in Panel D - Timepoint 0 (no incubation) BNPAGE_PanelD_time14d.jpg----------------&gt;&gt;BNPAGE (coomassie stained) original image of the gel presented in Panel D - 14 days incubation BNPAGE_PanelD_time48hrs.jpg----------------&gt;&gt;BNPAGE (coomassie stained) original image of the gel presented in Panel D - 48 hrs incubation BNPAGE_PanelD_time8d.jpg----------------&gt;&gt;BNPAGE (coomassie stained) original image of the gel presented in Panel D -8 days incubation Fig2_supp_fil_2_Annotated_gels.pdf----------------&gt;&gt;PDF files showing the uncropped Blue-NATIVE PAGE gel and blot shown in the figure with annotation Figure2_Figure_Supplement_3.zip: Figure2_Figure_Supplement_3_A.csv: Histogram A panel data. MW ranges and counts Figure2_Figure_Supplement_3_B.csv: Histogram B panel data. MW ranges and counts Figure2_Figure_Supplement_3_C.csv: Histogram C panel data. MW ranges and counts Figure_3.zip: zipped folder containing the following files Annotated_gels_Figure_3.pdf ----------------&gt;&gt; PDF files containing the annotated , uncropped pictures of all the agarose gel presented in the figure Cleavage_assay_with_GyrB.tif ----------------&gt;&gt; original picture in Tif format of the cleavage assays performed with various heterodimers in the presence of GyrB Cleavage_assay_without_GyrB.tif ----------------&gt;&gt; original picture in Tif format of the cleavage assays (DNA resolved with an agarose gel) performed with various heterodimers in the absence of GyrB Supercoiling_assay_with_GyrB.tif ----------------&gt;&gt; original picture in Tif format of the supercoiling assays (DNA resolved with an agarose gel) performed with various heterodimers in the presence of GyrB Supercoiling_assay_without_GyrB.tif ----------------&gt;&gt; original picture in Tif format of the cleavage assays (DNA resolved with an agarose gel) performed with various heterodimers in the absence of GyrB Figure_3_figure_supplement_1.zip: zipped folder containing the following files Annotated_gel_Fig3_supp_figure_1.pdf ----------------&gt;&gt; PDF file PDF files containing the annotated, uncropped pictures of all the agarose gel presented in the figure repeat1_Fig3_figure_supp_1.tif ----------------&gt;&gt; original picture of the agarose gel presented in the figure. repeat2and3_Fig_3_figure_supp_1.tif ----------------&gt;&gt; the experiment depicted in the figure was repeated three times. This is the original image for repeat 2 and 3 (not presented in the figure) Figure_3_figure_supplement_2.zip: zipped folder containing the following files 2018_07_05_gel1_Figure_3_fig_supp_2_a.tif ----------------&gt;&gt; original picture of the cleavage assays (analysed by agarose gel) presented in the A panel 2018_07_05_gel2_Figure_3_fig_supp_2_a_WITHOUT_GYRB.tif ----------------&gt;&gt; the cleavage assays presneted in panel A were reproduced without GyrB. This file contain the original image for these assays. 2019_09_14_Figure_3_figure_supplement_3_b.tif ----------------&gt;&gt; original picture of the cleavage assays (analysed by agarose gel) presented in the B panel Annotated_gel_Figure_3_figure_supplement_2.pdf ----------------&gt;&gt; PDF file PDF files containing the annotated, uncropped pictures of all the agarose gel presented in the figure, plus the assays performed without GyrB Figure_4.zip: zipped folder containing the following files Annotated_radiolabelling_Figure_4.pdf ----------------&gt;&gt; PDF file containing the uncropped autoradiogram presented in figure 4 with annotation Radiolabelling_Figure_4.tif ----------------&gt;&gt; original picture of the autoradiogram Figure_4_figure_supplement_1.zip: zipped folder containing the following files Annotated_gel_Figure_4_figure_supp_1.pdf ----------------&gt;&gt; PDF file containing the uncropped agarose gel presented in the figure with annotation. Figure_4_Supp_figure_1.tif ----------------&gt;&gt; original picture of the agarose gel Figure_5.zip: zipped folder containing the following files Annotated_gel_Figure_5.pdf ----------------&gt;&gt; PDF file containing the uncropped agarose gel presented in the figure with annotation. Figure_5.tif ----------------&gt;&gt; original picture of the agarose gel Figure_6.zip: zipped folder containing the following files Annotated_Gels_Figure_6.pdf ----------------&gt;&gt; PDF file containing the uncropped agarose gels presented in the figure with annotation. Figure_6_A.tif ----------------&gt;&gt; original picture of the agarose gel presented in panel A Figure_6_B.tif ----------------&gt;&gt; original picture of the agarose gel presented in panel B Figure_6_C.tif ----------------&gt;&gt; original picture of the agarose gel presented in panel C Figure_6_D.tif ----------------&gt;&gt; original picture of the agarose gel presented in panel D Without_GyrB_Figure_6_C.tif ----------------&gt;&gt; original picture for the assays presented in panel C and re-done without GyrB. Figure_6_figure_supplement_1.zip: zipped folder containing the following files 2019_11_20_1_F6_FIGURE_SUPP_1_A.tif ----------------&gt;&gt; original picture of the agarose gel presented in panel A (TOP, cleavage assays) 2019_11_20_2-1_F6_FIGURE_SUPP_1_B.tif ----------------&gt;&gt; original picture of the agarose gel presented in panel B (TOP, cleavage assays) 2019_11_20_3_F6_FIGURE_SUPP_1_A.tif ----------------&gt;&gt; original picture of the agarose gel presented in panel A (BOTTOM, supercoiling assays) 2019_11_20_4_F6_FIGURE_SUPP_1_B.tif ----------------&gt;&gt; original picture of the agarose gel presented in panel B (BOTTOM, supercoiling assays) Annotated_gels_figure_6_figure_supp_1.pdf ----------------&gt;&gt; PDF file containing the uncropped agarose gels presented in the figure with annotation. Figure_6_figure_supplement_2.zip: zipped folder containing the following files 2015_12_15_gel1_F6_FIGURE_SUPP_2_B.tif ----------------&gt;&gt; original picture of the agarose gel presented in panel B 2016_04_21_CQ1_F6_FIGURE_SUPP_2_C_WITH_CHLOROQUINE.tif ----------------&gt;&gt; original picture of the agarose gel ran in the presence of Chloroquine and presented in panel C 2016_04_21_F6_FIGURE_SUPP_2_C.tif ----------------&gt;&gt; original picture of the agarose gel ran in the absence of Chloroquine and presented in panel C Annotated_gel_Figure_6_supp_figure_2.pdf ----------------&gt;&gt; PDF file containing the uncropped agarose gels presented in the figure with annotation. binding_assay_wt_51C_2016_02_23_B_F6_FIGURE_SUPP_2_A.txt ----------------&gt;&gt; raw data for the SPR assay. Format is tab-separated value. Once plotted the identity of each trace becomes obvious, please refer to the figure. Figure_7.zip: zipped folder containing the following files Annotated_gels_Figure_7.pdf ----------------&gt;&gt; PDF file containing the uncropped agarose gels presented in the figure with annotation. Gel_BA_A_wild_type.tif ----------------&gt;&gt; original picture of the agarose gel for the BA.A wild-type heterodimer cleavage kinetic gel_BALLL_A_mutant.tif ----------------&gt;&gt; original picture of the agarose gel for the BA.A wild-type heterodimer cleavage kinetic Figure_8_figure_supplement_6.zip: ipped folder containing the following files Annotated_gel_Figure_8_figure_supplement_6.pdf ----------------&gt;&gt; PDF file containing the uncropped agarose gels presented in the figure with annotation. Figure_8_figure_supplement_6.tif ----------------&gt;&gt; original picture of the agarose gel presented in the picture.