Topoisomerase VI is a chirally-selective, preferential DNA decatenase
- Laboratory of Single Molecule Biophysics, National Heart, Lung and Blood Institute, National Institutes of Health, United States
- Department of Biochemistry and Metabolism, John Innes Centre, United Kingdom
Figures
Figure 1
Topo VI relaxation rate depends on the chirality of DNA supercoiling.
(A) Magnetic tweezers calibration curves for a 5 kb DNA duplex supercoiled under low (0.2 pN), medium (0.5 pN) and high (1.0 pN) force. DNA extension is plotted as a function of magnet turns. …
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Figure 1—source data 1
Source data is in the file Figure 1.
- https://cdn.elifesciences.org/articles/67021/elife-67021-fig1-data1-v2.csv
Figure 2
Topo VI relaxation rate depend on the level of DNA supercoiling.
(A) An example trace of 0.75 nM topo VI fully relaxing negative supercoils in a 5 kb DNA duplex at a force of 0.4 pN, at 21 °C. DNA extension is plotted against time. The abrupt decreases in …
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Figure 2—source data 1
Source data is in the file Figure 2.
- https://cdn.elifesciences.org/articles/67021/elife-67021-fig2-data1-v2.csv
Figure 3 with 1 supplement
ATPase activity and DNA binding of topo VI are stimulated by supercoiled DNA.
(A) The ATPase activity of topo VI over time, measured using radioactive ATP. Assays were performed at 21 °C, using 1 μM topo VI, 430 nM pBR322* that was negatively-supercoiled (blue), linear …
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Figure 3—source data 1
Source data is in the file Figure 3.
- https://cdn.elifesciences.org/articles/67021/elife-67021-fig3-data1-v2.csv
Figure 3—figure supplement 1
ATPase activity of M. mazei topo VI with different DNA species.
ATPase rates were measured using a PK/LDH linked assay (Feng et al., 2021) with 1.2 µM topo VI in the presence of 177 nM DNA at 37 °C.
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Figure 3—figure supplement 1—source data 1
Source data is in the file Figure 3—figure supplement 1.
- https://cdn.elifesciences.org/articles/67021/elife-67021-fig3-figsupp1-data1-v2.csv
Figure 4 with 3 supplements
Topo VI activity on braided DNA substrates.
(A) Calibration curve for a DNA braid formed from two 5 kb DNA duplexes tethered to a single magnetic bead. DNA extension is plotted as a function of magnet turns. Negative magnet-turn values …
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Figure 4—source data 1
Source data is in the file Figure 4.
- https://cdn.elifesciences.org/articles/67021/elife-67021-fig4-data1-v2.csv
Figure 4—figure supplement 1
Additional examples of T-test fits to braid relaxation data.
Extension versus time data for topo VI relaxing braided DNA (red lines) and the T-test based fit to the data (black lines). The sign of the braiding and the concentration of topo VI are indicated on …
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Figure 4—figure supplement 1—source data 1
Source data is in the file Figure 4—figure supplement 1.
- https://cdn.elifesciences.org/articles/67021/elife-67021-fig4-figsupp1-data1-v2.xlsx
Figure 4—figure supplement 2
Comparison of the experimental braid relaxation data with a purely distributive relaxation model via simulations.
To test the possibility that the braid relaxation data are consistent with purely distributive relaxation by topo VI, we performed simulations of distributive braid relaxation with average rates …
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Figure 4—figure supplement 2—source data 1
Source data is in the file Figure 4—figure supplement 2.
- https://cdn.elifesciences.org/articles/67021/elife-67021-fig4-figsupp2-data1-v2.xlsx
Figure 4—figure supplement 3
Examples of T-test based fits to simulated braid relaxation data.
Simulated DNA extension versus time for distributive relaxation (grey line) along with the T-test fit (black line) and the simulated extension in the absence of noise (green dashed line) at average …
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Figure 4—figure supplement 3—source data 1
Source data is in the file Figure 4—figure supplement 3.
- https://cdn.elifesciences.org/articles/67021/elife-67021-fig4-figsupp3-data1-v2.xlsx
Figure 5
Agarose gel-based assay of DNA decatenation and relaxation by Methanosarcina mazei topo VI (MmT6).
On the left, a singly catenated (depicted by the linked green and orange circles), negatively supercoiled plasmid substrate is decatenated by 0.1–20 nM topo VI. The catenated, supercoiled plasmids …
Figure 6 with 1 supplement
Calculated average DNA-crossing angles for supercoils and braids, as a function of force.
The average DNA-crossing angle in positive and negative supercoils (+ sc [ink] and -sc [light blue], respectively) were calculated as described in Neukirch and Marko, 2011. The temperature was 293 …
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Figure 6—source data 1
Source data is in the file Figure 6.
- https://cdn.elifesciences.org/articles/67021/elife-67021-fig6-data1-v2.csv
Figure 6—figure supplement 1
Effect of force on the braid unlinking and supercoil relaxation activity of topo VI.
(A) The average rate of positive (red, N tethers across all data points = 28) and negative (blue, N tethers across all data points = 22) braid unlinking (events min–1) (± SEM), plotted against …
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Figure 6—figure supplement 1—source data 1
Source data is in the file Figure 6—figure supplement 1.
- https://cdn.elifesciences.org/articles/67021/elife-67021-fig6-figsupp1-data1-v2.csv
Figure 7 with 1 supplement
Topo VI unlinking single DNA crossings.
(A) Single crossing assay schematic with DNA crossing geometry for positive (left-handed, red) and negative (right-handed, blue) DNA writhe. One 360° clockwise magnet rotation imparts a positive …
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Figure 7—source data 1
Source data is in the file Figure 7.
- https://cdn.elifesciences.org/articles/67021/elife-67021-fig7-data1-v2.csv
Figure 7—figure supplement 1
Braid tether calibration and geometric fit.
(A) The DNA extension of a 3 kb double tether, plotted as a function of magnet rotations and fitted to a geometric function (Neuman et al., 2009). Where L is the measured DNA extension, L0 is the …
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Figure 7—figure supplement 1—source data 1
Source data is in the file Figure 7—figure supplement 1.
- https://cdn.elifesciences.org/articles/67021/elife-67021-fig7-figsupp1-data1-v2.csv
Figure 8 with 2 supplements
Preferred DNA crossing angle measurements for topo VI.
(A) Crossing angle probability distributions for single positive (PL(α), red) and negative (PR(α), blue) crossings, from Monte Carlo (MC) simulations for the tether geometry and force displayed in Fi…
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Figure 8—source data 1
Source data is in the file Figure 8.
- https://cdn.elifesciences.org/articles/67021/elife-67021-fig8-data1-v2.csv
Figure 8—figure supplement 1
Estimate of the preferred crossing angle for topo VI from plasmid relaxation ATPase measurements.
Comparison between the ATPase rate of Topo VI relaxing negatively versus positively supercoiled DNA and the crossing angle distributions of negatively- and positively-supercoiled DNA estimated from …
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Figure 8—figure supplement 1—source data 1
Source data is in the file Figure 8—figure supplement 1.
- https://cdn.elifesciences.org/articles/67021/elife-67021-fig8-figsupp1-data1-v2.csv
Figure 8—figure supplement 2
Comparisons of crossing angle probability distributions among single-crossings that are relaxed and not relaxed by topo VI.
(A) Crossing angle probability distributions from MC simulations for single-crossing geometries that were relaxed by topo VI (solid lines) and one single-crossing geometry that was not relaxed by …
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Figure 8—figure supplement 2—source data 1
Source data is in the file Figure 8—figure supplement 2.
- https://cdn.elifesciences.org/articles/67021/elife-67021-fig8-figsupp2-data1-v2.csv
Figure 9
Model for chirality-dependent topo VI activity.
Unbound topo VI (1) binds a G- and T-segment in the presence of ATP (2), leading to G-segment cleavage and T-segment strand-passage. The rate at which strand passage occurs is sensitive to the …
Author response image 1
Average preferred crossing angle for topo VI obtained from comparisons among all right handed crossings and all left handed crossings for single-crossing relaxation experiments.
Each point represents the average crossing angle obtained by comparing the relative relaxation times and simulated DNA crossing angle distributions of 26 single-crossing measurements (13 of each …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Methanosarcina mazei) | Top6A | Gift from James Berger, Johns Hopkins University | NCBI Gene ID: 1480760 | |
| Gene (Methanosarcina mazei) | Top6B | Gift from James Berger, Johns Hopkins University | NCBI Gene ID: 1480759 | |
| Strain, strain background (E. coli) | Rosetta 2 (pLysS) | Novagen | ||
| Genetic reagent (E. coli) | pBR322* | Inspiralis | ||
| Genetic reagent (E. coli) | pET28a | EMD Millipore | CAT#:69,865 | |
| Genetic reagent (E. coli) | pBlueScript II KS(+) | Agilent | CAT#:212,207 | |
| Recombinant DNA reagent | Top6AB dual expression vector | PMID:17603498 | Corbett et al., 2007 | |
| Antibody | Anti-digoxigenin(SheepPolyclonal) | Roche | Roche Cat# 11333089001, RRID:AB_514496 | Reconstituted in 1 x Phosphatebuffered saline (0.6 µg) |
| Commercial assay or kit | PCR DNA purification kit | Qiagen | Qiagen Cat. #: 28,104 | |
| Other | Streptavidin coated magnetic beads (ø: 1 and 2.8 µm) | Invitrogen | Invitrogen Cat. #: 65,602 and 65,305 | |
| Chemical compound, drug | Phusion high-fidelity DNA polymerase | New England Biolabs | NEB Cat. #: M0530 | |
| Chemical compound, drug | T4 DNA ligase | Promega | Promega Cat. #: M1801 | |
| Chemical compound, drug | BsaI-HF | New England Biolabs | NEB Cat. #: R3535 | |
| Chemical compound, drug | Biotin-16-dUTP | Roche | Sigma Cat#:11093070910 | |
| Chemical compound, drug | Digoxigenin-11-dUTP | Roche | Sigma Cat#:11558706910 | |
| Sequence-based reagent | 5 kb DNA supercoil primer1 | Eurofin Genomics Seol and Neuman, 2011a | 5'- GCT GGG TCT CGG TTGTTC CCT TTA GTG AGG GTT AAT TG | |
| Sequence-based reagent | 5 kb DNA supercoil primer2 | Eurofin Genomics Seol and Neuman, 2011a | 5'- GCT GGG TCT CGT GGT TTC CCTTTA GTG AGG GTT AAT TG | |
| Sequence-based reagent | 3 kb DNA braid primer1 | Eurofin Genomics | 5’(2 x)biotin-GCTGGGTCTCGGTTGGAACTGCGACT GGATAGG | |
| Sequence-based reagent | 3 kb DNA braid primer 2 | Eurofin Genomics | 5' (3 x) digoxigenin-GCTGGGTCTCGGTTGGATTACGCCA GTTGTACG | |
| Sequence-based reagent | 5 kb DNA braid primer1 | Eurofin Genomics | 5’(2 x)biotin-CTTCCGCTTCCTCGCTCACTGACTC | |
| Sequence-based reagent | 5 kb DNA braid primer 2 | Eurofin Genomics | 5' (3 x) digoxigenin-CTGTTCATCCGCGTCCAGCTCGTTG | |
| Sequence-based reagent | Bio/Dig labelledPrimer1 | Eurofin Genomics Seol and Neuman, 2011a | 5’-GGA CCT GCT TTC GTT GTG GCG TAA TCA TGG TCA TAG | |
| Sequence-based reagent | Bio/Dig labelledPrimer2 | Eurofin Genomics Seol and Neuman, 2011a | 5'- GGG TCT CGT GGT TTA TAG TCC TGT CGG GTT TC | |
| Software, algorithm | LabVIEW, Instrument control software | National Instruments | NI Cat. #: 776678–35 | |
| Software, algorithm | Igor Pro 7,Data analysis | WaveMetrics | PMID:28069956 | |
| Software, algorithm | ImageJ, Data analysis | National Institutes of Health | ||
| Chemical compound, drug | Adenosine triphosphate (ATP) | MilliporeSigma | A2383 | |
| Chemical compound, drug | Nicotinamide adenine dinucleotide (NADH) | MilliporeSigma | 10107735001 | |
| Chemical compound, drug | Pyruvate Kinase/Lactic Dehydrogenase (PK/LDH) | MilliporeSigma | P0294 | |
| Chemical compound, drug | Phosphoenol-pyruvate (PEP) | MilliporeSigma | 10108294001 | |
| Other | Plate reader | BMG LabTech | CLARIOstar Plus | Used for the PK/LDH-coupled ATPase assay. |
| Software, algorithm | Microsoft Excel | RRID:SCR_016137 | Used for data analysis for the PK/LDH-coupled ATPase assay. | |
| Software, algorithm | LAMMPS | https://www.lammps.org/ | Used for Molecular Dynamics Simulations | |
| Software, algorithm | MATLAB | MathWorks | Used for analyses of Molecular Dynamics Simulations |
