Topoisomerase Assays

John L. Nitiss1, Eroica Soans2, Anna Rogojina1, Aman Seth2, Margarita Mishina2

1 Biopharmaceutical Sciences Department, University of Illinois College of Pharmacy at Rockford, Rockford, Illinois, 2 Molecular Pharmacology Department, St. Jude Children's Research Hospital, Memphis, Tennessee
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 3.3
DOI:  10.1002/0471141755.ph0303s57
Online Posting Date:  June, 2012
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Abstract

Topoisomerases are nuclear enzymes that play essential roles in DNA replication, transcription, chromosome segregation, and recombination. All cells have two major forms of topoisomerases: type I enzymes, which make single‐stranded cuts in DNA, and type II enzymes, which cut and pass double‐stranded DNA. DNA topoisomerases are important targets of approved and experimental anti‐cancer agents. The protocols described in this unit are for assays used to assess new chemical entities for their ability to inhibit both forms of DNA topoisomerase. Included are an in vitro assay for topoisomerase I activity based on relaxation of supercoiled DNA, and an assay for topoisomerase II based on the decatenation of double‐stranded DNA. The preparation of mammalian cell extracts for assaying topoisomerase activity is described, along with a protocol for an ICE assay to examine topoisomerase covalent complexes in vivo, and an assay for measuring DNA cleavage in vitro. Curr. Protoc. Pharmacol. 57:3.3.1‐3.3.27. © 2012 by John Wiley & Sons, Inc.

Keywords: topoisomerase; topoisomerase I; topoisomerase II; camptothecin; etoposide; topoisomerase poison

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Assay of Topoisomerase I Activity
  • Basic Protocol 2: Assay of Topoisomerase II Activity
  • Support Protocol 1: Preparation of Mammalian Cell Extracts for Assaying Topoisomerase Activity
  • Basic Protocol 3: In Vivo Determination of Topoisomerase Covalent Complexes using the In‐Vivo Complex of Enzyme (ICE) Assay
  • Support Protocol 2: Preparation of Mouse Tissue for In Vivo Determination of Topoisomerase Covalent Complexes using the In Vivo Complex of Enzyme (ICE) Assay
  • Basic Protocol 4: Determination of DNA Cleavage by Purified Topoisomerase I
  • Basic Protocol 5: Determination of Inhibitor Effects on DNA Cleavage by Topoisomerase II using a Plasmid Linearization Assay
  • Alternate Protocol 1: Gel Electrophoresis Determination of Topoisomerase II Cleavage
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Assay of Topoisomerase I Activity

  Materials
  • 10× topoisomerase I reaction buffer (see recipe)
  • Substrate: plasmid DNA
  • Purified topoisomerase I or cell extract (see protocol 3)
  • 5× loading dye (see recipe)
  • 0.8% agarose gel (Voytas, )
  • Additional reagents and equipment for agarose gel electrophoresis, ethidium bromide staining, and gel photography (Voytas, )

Basic Protocol 2: Assay of Topoisomerase II Activity

  Materials
  • 10× topoisomerase II reaction buffer (see recipe)
  • Substrate: kinetoplast DNA (Topogen, http://www.topogen.com)
  • Purified topoisomerase II (see Critical Parameters) or cell extract (see protocol 3)
  • 5× loading dye (see recipe)
  • 0.8% agarose gel (Voytas, )
  • Additional reagents and equipment for agarose gel electrophoresis, ethidium bromide staining, and gel photography (Voytas, )

Support Protocol 1: Preparation of Mammalian Cell Extracts for Assaying Topoisomerase Activity

  Materials
  • Cells of interest growing in tissue culture: e.g., HeLa (ATCC #CCL‐2.2) or K562 (ATCC #HB‐84)
  • Phosphate‐buffered saline (PBS; see recipe), room temperature and ice cold
  • PBS (see recipe) containing 5 mM EDTA
  • Low‐salt extraction buffer (see recipe), ice cold
  • High‐salt extraction buffer: low‐salt extraction buffer containing 0.35 M KCl
  • 15‐ml centrifuge tubes
  • Hemacytometer (or automated cell counter)
  • Refrigerated centrifuge
  • Dounce homogenizer with loose pestle, precooled
  • Phase‐contrast microscope
  • Additional reagents and equipment for measuring protein concentration ( appendix 3A)

Basic Protocol 3: In Vivo Determination of Topoisomerase Covalent Complexes using the In‐Vivo Complex of Enzyme (ICE) Assay

  Materials
  • Cells of interest: e.g., HeLa (ATCC #CCL‐2.2) or K562 (ATCC #HB‐84)
  • Test compound(s)
  • 1% (w/v) Sarkosyl in 1× TE buffer (see appendix 2A for TE buffer)
  • CsCl solution: add 75 g of CsCl (molecular biology grade) to 50 ml H 2O; if necessary, warm to completely dissolve the CsCl (stable indefinitely at room temperature)
  • 70% ethanol
  • 1× TE buffer, pH 7.5 ( appendix 2A)
  • 25 mM sodium phosphate buffer, pH 6.5 ( appendix 2A)
  • PBS‐T: 1× phosphate‐buffered saline (see recipe) containing 0.01% (v/v) Tween 20
  • Blocking solution: 5% (w/v) instant non‐fat dry milk (Nestle, Carnation) in PBS‐T
  • Primary antibody, e.g.:
    • Rabbit anti‐ human TOPIIα IgG antibody (Bethyl Laboratories, cat. no. BL 983)
    • Mouse anti‐human TOPIIβ IgG antibody (BD Transduction Laboratories, cat. no. 611493)
  • Secondary antibodies:
    • For rabbit primary antibody: ECL anti‐rabbit IgG, horse peroxidase‐linked antibody from donkey (Amersham, cat. no. NA934V)
    • For mouse primary antibody: ECL anti‐mouse IgG, horse peroxidase‐linked antibody from donkey (Amersham, cat. no. NA934V)
  • Amersham ECL Plus Western Blotting Detection Reagents (GE Healthcare, cat. no. RPN2132)
  • 100‐mm culture dishes
  • 14‐ml polypropylene round‐bottom tubes
  • 1‐ml latex‐free syringe with 25‐G 5/8 in. needle (Becton Dickinson)
  • 3‐ml latex‐free syringe with a 16‐G 1/ 2 in. precision‐glide needle (Becton Dickinson)
  • OptiSeal tubes for ultracentrifugation (Beckman Coulter)
  • Beckman ultracentrifuge with NVt90 rotor, or equivalent
  • Sharp blade
  • 65°C water bath
  • Trans Blot nitrocellulose membrane (BioRad, cat. no. 162‐0115)
  • Slot‐format filter paper (BioRad, cat. no. 162‐0161)
  • Slot blotting apparatus (Bio‐Dot SF apparatus, BioRad, cat. no. 170‐6542)
  • Hoefer Red orbital shaker, or equivalent
  • Additional reagents and equipment for quantitation of DNA (Desjardins and Conklin, ; Gallagher, )

Support Protocol 2: Preparation of Mouse Tissue for In Vivo Determination of Topoisomerase Covalent Complexes using the In Vivo Complex of Enzyme (ICE) Assay

  Materials
  • Mouse tissue sample
  • 1% (w/v) Sarkosyl in 1× TE buffer (see appendix 2A for TE buffer)
  • 2‐ml tissue grinder (Wheaton)
  • 14‐ml polypropylene round‐bottom tube
  • 1‐ml latex‐free syringe with 25‐G 5/8 in. needle (Becton Dickinson)
  • Additional reagents and equipment for ICE Assay ( protocol 4)

Basic Protocol 4: Determination of DNA Cleavage by Purified Topoisomerase I

  Materials
  • Oligonucleotides (custom‐synthesized):
    • 5′‐GATCTAAAAGACTTGGAAAAATTTTTAAAAAAGATC‐3′ (upper strand)
    • 5′‐GATCTTTTTTAAAAATTTTTCCAAGTCTTTTAGATC‐3′ (lower strand)
  • [α‐32P]cordycepin 5000 Ci/mmol (Perkin‐Elmer Product BLU26250UC)
  • Terminal deoxynucleotidyl transferase (TdT) labeling kit (Stratagene)
  • 10× topoisomerase I cleavage buffer (see recipe)
  • 50 U/µl purified topoisomerase I (Topogen; also see Critical Parameters; 1 U topoisomerase I is the quantity of enzyme that will relax 200 ng pUC18 DNA in 30 min)
  • Compounds to be tested
  • 5% (w/v) SDS
  • Formamide loading buffer (see recipe)
  • 30‐ to 40‐cm denaturing 16% polyacrylamide/7 M urea sequencing gel prepared as described in Slatko and Albright ( ); also see Albright and Slatko ( ); electrophoresis of DNA on sequencing gels requires a large vertical gel apparatus, a high‐voltage power supply, and a darkroom for developing autoradiograms (or a phosphoimager)
  • Sephadex G‐25 spin columns (GE Healthcare)
  • Scintillation vials, scintillation fluid, and counter
  • 95°C heating block
  • 400‐µl microcentrifuge tubes
  • 25°C water bath
  • Whatman 3MM filter paper
  • Additional reagents and equipment for sequencing by denaturing polyacrylamide gel electrophoresis (Slatko and Albright, ; Albright and Slatko, )
CAUTION: Radioactive materials require special handling; all supernatants must be considered radioactive waste and disposed of appropriately.

Basic Protocol 5: Determination of Inhibitor Effects on DNA Cleavage by Topoisomerase II using a Plasmid Linearization Assay

  Materials
  • 10× topoisomerase II reaction buffer
  • Supercoiled plasmid DNA (see Critical Parameters)
  • Test compound(s)
  • Positive control (e.g., etoposide, mAMSA)
  • 4 to 20 U/µl purified topoisomerase II (see Critical Parameters)
  • 10% (w/v) SDS
  • 250 mM tetrasodium EDTA, pH 8.0
  • 4 mg/ml stock solution of proteinase K: dilute to 0.8 mg/ml before use
  • 20 mM ATP (diluted with H 2O from purchased stock solution; store up to 6 months in small aliquots at −20°C)
  • 5× loading dye (see recipe)
  • 0.8% agarose gel (Voytas, )
  • Restriction enzyme appropriate for cutting plasmid once, to linearize plasmid
  • 10 mg/ml ethidium bromide stock solution: store at room temperature, protected from light
  • Gel densitometer
  • Additional reagents and equipment for performing agarose gel electrophoresis, ethidium bromide staining, and gel photography (Voytas, )

Alternate Protocol 1: Gel Electrophoresis Determination of Topoisomerase II Cleavage

  Materials
  • Substrate: plasmid DNA (e.g., pUC18 or pBluescript)
  • EcoRI restriction endonuclease (additional restriction endonuclease, e.g., BamHI is optional)
  • Formamide loading buffer (see recipe)
  • 0.8% agarose gels (Voytas, )
  • TE buffer, pH 8.0 ( appendix 2A)
  • 5 mM dCTP, dGTP, and dTTP
  • 5 U/µl Klenow fragment of DNA polymerase I and 10× Klenow buffer
  • 10 mCi/ml [α‐32P] dATP (800 Ci/mmol; Perkin‐Elmer)
  • 10× topoisomerase II reaction buffer (see recipe)
  • Compounds to be tested
  • 20 to 40 U/µl purified topoisomerase II
  • 5% (w/v) SDS containing 1 mg/ml proteinase K
  • 5× loading dye (see recipe)
  • Sephadex G‐25 spin column (GE Healthcare)
  • Additional reagents and equipment for agarose gel electrophoresis (Voytas, ), purification of DNA by phenol/chloroform/isoamyl alcohol extraction and ethanol precipitation ( appendix 3C), and autoradiography (Voytas and Ke, )
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Figures

Videos

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Key References
   Bjornsti, M.A. and Osheroff, N (eds.). DNA Topoisomerase Protocols: Volume I: DNA Topology and Enzymes Methods in Molecular Biology, Vol. 94, 1999 Print ISBN: 978‐0‐89603‐444‐0. Humana Press, Totowa, N.J.
  Detailed protocols for many different aspects of DNA topoisomerases.
   Bjornsti, M.A. and Osheroff, N (eds.). DNA Topoisomerase Protocols: Volume II: Enzymology and Drugs Methods in Molecular Biology, Vol. 95, 1999 Print ISBN: 978‐0‐89603‐512‐6. Humana Press, Totowa, N.J.
  Introduction and current status of the biology and biochemistry of DNA topoisomerases.
   Nitiss, J.L. 2009a. See above.
  Introduction to drugs targeting DNA topoisomerases
   Wang, J.C. 2002. See above.
   Nitiss, J.L. 2009. See above.
   Pommier, Y. 2006. See above.
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