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E coli topoisomerase 1

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E. coli Topoisomerase I is an enzyme that catalyzes the relaxation of supercoiled DNA. It introduces transient single-strand breaks in DNA to relieve torsional stress.

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Lab products found in correlation

Cutsmart buffer, by New England Biolabs (1 mentions) Bamhi hf, by New England Biolabs (1 mentions) Chitin beads, by New England Biolabs (1 mentions) Terminal transferase, by Roche (1 mentions) Nbt bcip solution, by Roche (1 mentions) Anti dig antibody, by Roche (1 mentions) Ni nta matrix, by Qiagen (1 mentions) Genomic dna isolation kit, by HiMedia (1 mentions) Dig labeled ddutp, by Roche (1 mentions) Sybr safe, by Thermo Fisher Scientific (1 mentions) Calf thymus topoisomerase 1, by Takara Bio (1 mentions) Proteinase k, by Thermo Fisher Scientific (1 mentions) Chemidoc mp system, by Bio-Rad (1 mentions)

Close spelling variants of this product

Topoisomerase I (E. coli) (M0301) Topoisomerase I

4 protocols using e coli topoisomerase 1

1

DNA Isolation and Topoisomerase Analysis

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Cells were washed once with PBS and then resuspended in lysis buffer (75 mM NaCl, 50 mM EDTA, 20 mM HEPES (pH 7.8), 0.5% (w/v) SDS and 0.2 mg/ml proteinase K). DNA was isolated by sequential phenol and chloroform extractions and resuspended in TE (pH 8). DNA (2.5 μg) was separated on 0.4% agarose gels, without ethidium bromide, at 35 V for 22 h. As controls, 1 μg of DNA from wild‐type U2OS Flp‐In cells was treated with 10 U of BamHI‐HF (NEB) or 5 U of E. coli Topoisomerase I (NEB) for 30 min at 37°C in 1 × CutSmart buffer (NEB), before being loaded on gels alongside uncut DNA as above. After electrophoresis, gels were prepared for Southern blotting as above for restricted samples.
Erdinc D., Rodríguez‐Luis A., Fassad M.R., Mackenzie S., Watson C.M., Valenzuela S., Xie X., Menger K.E., Sergeant K., Craig K., Hopton S., Falkous G., Poulton J., Garcia‐Moreno H., Giunti P., de Moura Aschoff C.A., Morales Saute J.A., Kirby A.J., Toro C., Wolfe L., Novacic D., Greenbaum L., Eliyahu A., Barel O., Anikster Y., McFarland R., Gorman G.S., Schaefer A.M., Gustafsson C.M., Taylor R.W., Falkenberg M, & Nicholls T.J. (2023). Pathological variants in TOP3A cause distinct disorders of mitochondrial and nuclear genome stability. EMBO Molecular Medicine, 15(5), e16775.
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2

Biochemical Toolkit for DNA Manipulation

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Restriction enzymes, vectors (pTWIN1 & pMBX10), DNA (ØX virion, ØX dsDNA and M13mp18 dsDNA), E. coli Topoisomerase I and chitin beads were obtained from New England Biolabs, UK. Q-Sepharose, Sephadex 50 columns, terminal transferase, Dig labeled ddUTP, anti-Dig antibody and NBT-BCIP solution were obtained from Roche Life Sciences, UK. Ni–NTA matrix was obtained from Qiagen, Germany. IPTG, ATP, phosphocreatine, phosphocreatine kinase and E. coli Ssb protein were obtained from Sigma–Aldrich India. Bacterial growth medium component were obtained from BD and Co., India. Oligo dT50 was obtained from MWG Biotech, India. Genomic DNA isolation kit was obtained from Hi-media Laboratories, India.
Ujaoney A.K., Basu B., Muniyappa K, & Apte S.K. (2015). Functional roles of N-terminal and C-terminal domains in the overall activity of a novel single-stranded DNA binding protein of Deinococcus radiodurans. FEBS Open Bio, 5, 378-387.
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3

Plasmid DNA Supercoiling Assay

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Negatively supercoiled pRS306 plasmid DNA was prepared by standard CsCl gradient purification. Relaxed covalently-closed circular (rCCC) plasmid DNA was prepared from negatively supercoiled plasmid DNA by treating with 1.33 units E. coli topoisomerase I (New England Biolabs) per microgram plasmid DNA at 37 °C for 30 minutes followed by phenol-chloroform extraction and ethanol precipitation. One-hundred and fifty nanograms of negatively supercoiled or rCCC plasmid were loaded along with linear DNA size markers in immediately adjacent lanes into a 0.9% agarose 1X TAE gel, with 4% DMSO ± 10 μM compound present throughout the gel and the running buffer. The gel was run at 5 V/cm until the xylene cyanol FF dye had migrated 5 cm from the well, stained with ethidium bromide, and photographed under UV transillumination. Densitometry traces were obtained for each lane using ImageJ, and intercalation scores were calculated from the lane loaded with rCCC plasmid as the median distance migrated by topoisomers for each compound relative to the DMSO-only control from the same experiment.
Budke B., Tueckmantel W., Miles K., Kozikowski A.P, & Connell P.P. (2019). Optimization of Drug Candidates that Inhibit the D-loop Activity of RAD51. ChemMedChem, 14(10), 1031-1040.
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4

DNA Supercoiling Assay with HU Proteins

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DNA supercoiling assays were performed as described by Guo and Adhya (2007) (link). Briefly, plasmid pCX eGFP was relaxed with E. coli topoisomerase I (New England Biolabs) according to manufacturer’s instructions. Then, 100 ng of relaxed DNA was prepared in 1× topoisomerase buffer (TaKaRa Bio), 0.01% BSA and incubated with 0.5 μg of HUβ2 or HUβ(K86ac)2 in a 10 μL reaction. The reaction was incubated at 37°C for 30 min. Then, 7 units of calf thymus topoisomerase I (TaKaRa Bio) was added to each reaction and the reactions incubated at 37°C for 2 h. 10 μg of proteinase K (Invitrogen) was added to each reaction, which were incubated at 37°C for 30 min. Then, 5× DNA loading dye (Qiagen, #239901) was added to each reaction and the samples were analyzed on 0.8% agarose in 1× TBE buffer and electrophoresed at 150 V for 90 min. Post-run, the gel was stained with SybrSafe (Invitrogen) according to manufacturer’s instructions and imaged on a Bio-Rad ChemiDoc MP system.
Barlow V.L, & Tsai Y.H. (2022). Acetylation at Lysine 86 of Escherichia coli HUβ Modulates the DNA-Binding Capability of the Protein. Frontiers in Microbiology, 12, 809030.
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