Replication Labeling with Halogenated Thymidine Analogs

Tomoki Yokochi1, David M. Gilbert1

1 Department of Biological Science Florida State University Tallahassee, Florida
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 22.10
DOI:  10.1002/0471143030.cb2210s35
Online Posting Date:  June, 2007
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Abstract

In this unit, several basic protocols to identify sites of DNA replication utilizing incorporation of halogenated thymidine analogs into DNA, followed by immunofluorescent imaging are described. Antibodies specific for halogenated thymidine analogs such as bromodeoxyuridine (BrdU), chlorodeoxyuridine (CldU), and iododeoxyuridine (IdU) can provide a rapid, nonhazardous, and sensitive method for detecting DNA replication in single cells, in a manner analogous to the traditional use of tritiated thymidine. In combination with different techniques to prepare the DNA template, a variety of DNA replication–related events can be examined by conventional fluorescence‐microscopic approaches. Because origin firing and the progression of replication forks are regulated in the context of subnuclear compartments through protein‐protein interactions, chromatin modifications, and subnuclear localization of replication clusters, visualizing replication foci significantly facilitates understanding of nuclear dynamics during S‐phase.

Keywords: DNA replication; mammalian cells; 5‐Bromo‐2′‐deoxyuridine (BrdU); halogenated thymidine analogs; DNA fiber

     
 
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Table of Contents

  • Basic Protocol 1: Visualizing DNA Replication Sites with Bromodeoxyuridine (Replication Labeling)
  • Basic Protocol 2: Combination of Replication Labeling and Fluorescence in Situ Hybridization (Fish)
  • Basic Protocol 3: Dual Replication Labeling with Chloro‐ and Iododeoxyuridine
  • Basic Protocol 4: Replication Labeling of DNA Fibers
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Visualizing DNA Replication Sites with Bromodeoxyuridine (Replication Labeling)

  Materials
  • Adherent tissue culture cells and appropriate medium
  • 10 mg/ml bromodeoxyuridine (BrdU) stock in sterile tissue culture–grade water (store indefinitely at −20°C)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • 2% (w/v) paraformaldehyde in PBS
  • 0.4% (v/v) Triton X‐100
  • 1.5 N HCl
  • 5% (w/v) BSA in PBST (0.5% v/v Tween 20 in PBS)
  • Primary antibody: monoclonal mouse anti‐BrdU antibody (Becton Dickinson)
  • Secondary antibody: Alexa Fluor 495–conjugated goat anti‐mouse antibody (Molecular Probes)
  • PBST: 0.5% (v/v) Tween 20 in PBS
  • 1 µg/ml DAPI in Vectashield (Vector Labs) mounting medium
  • 100‐mm tissue culture dishes for plating cells on coverslips
  • 12‐mm‐diameter round glass coverslips, sterilized by flaming after dipping in 70% ethanol or by autoclaving
  • 24‐well tissue culture plate
  • Glass microscope slides
  • Clear nail polish
  • Fluorescence microscope (unit 4.2) with appropriate color filters for fluorophores used, e.g., blue (DAPI), green (FITC, Alexa Fluor 488), and red (e.g., Texas Red, Alexa Fluor 594)
  • Additional reagents and equipment for cell culture (unit 1.1) and fluorescence microscopy (unit 4.2)

Basic Protocol 2: Combination of Replication Labeling and Fluorescence in Situ Hybridization (Fish)

  Materials
  • Adherent tissue culture cells and appropriate medium
  • 10 mg/ml bromodeoxyuridine (BrdU) stock (store indefinitely at −20°C)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Trypsin solution (see recipe)
  • Fixative: 3:1 (v/v) methanol/acetic acid (prepare fresh and store on ice)
  • 75 mM KCl
  • 0.0625 µg/µl probe DNA sequence in TE buffer (see appendix 2A for TE buffer)
  • Roche Nick Translation Mix (for digoxigenin or biotin, depending on desired labeling
  • 0.5 mM disodium EDTA, pH 8.0
  • Hybridization mix (see recipe)
  • 2× SSC ( appendix 2A) containing 0.1 mg/ml RNaseA
  • 2× and 0.1× SSC ( appendix 2A)
  • 70%, 90%, and 100% (v/v) ethanol
  • Clear nail polish
  • 2× SSC ( appendix 2A)/70% (v/v) formamide
  • 2× SSC ( appendix 2A)/50% (v/v) formamide
  • SSCT: 4× SSC ( appendix 2A)/0.1% (v/v) Tween 20
  • Blocking solution: SSCT (see above) containing 5% (w/v) nonfat dry milk
  • Primary antibody solution: cocktail in blocking solution of sheep anti‐digoxigenin, FITC‐conjugated (Molecular Probes) and mouse anti‐BrdU (Becton Dickinson), each at a dilution of 1:10
  • Secondary antibody solution: cocktail in blocking solution of rabbit anti–sheep IgG, FITC‐conjugated (Vector Labs), diluted 1:10, and goat anti–mouse IgG, Alexa Fluor 594–conjugated (Molecular Probes), diluted 1:500
  • Vectashield mounting medium (Vector Labs)
  • 100‐mm tissue culture dishes
  • 15‐ml conical centrifuge tubes
  • Centrifuge
  • Glass microscope slides
  • 15°C water bath (kept in cold room)
  • 45°, 65°, and 80°C water bath
  • Coplin jars
  • Vacuum desiccator with desiccant (e.g., silica gel)
  • 18 × 18–mm square glass coverslips, sterile
  • Slide warmer (Precision Scientific)
  • Humidified chamber (e.g., covered Tupperware container with moistened paper towels on the bottom)
  • 22 × 40–mm glass coverslips
  • Fluorescence microscope (unit 4.2) with appropriate color filters for fluorophores used, e.g., blue (DAPI), green (FITC, Alexa Fluor 488), and red (e.g., Texas Red, Alexa Fluor 594)
  • Additional reagents and equipment for cell culture technique including counting cells (unit 1.1) and fluorescence microscopy (unit 4.2)

Basic Protocol 3: Dual Replication Labeling with Chloro‐ and Iododeoxyuridine

  Materials
  • Cell culture on 12‐mm round coverslips ( protocol 1) in culture dish
  • 10 mg/ml chlorodeoxyuridine (CldU) stock solution (store indefinitely at −20°C)
  • 10 mg/ml iododeoxyuridine (IdU) stock solution (store indefinitely at −20°C)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • 70% (v/v) ethanol
  • 100% methanol
  • 1.5 N HCl
  • PBST (0.5% v/v Tween 20 in PBS) containing 5% (w/v) BSA
  • PBST: 0.5% (v/v) Tween 20 in PBS
  • Primary antibody for CldU: rat anti‐BrdU (Accurate Chemical)
  • Secondary antibody for CldU: FITC‐conjugated goat anti‐rat Ig (Molecular Probes)
  • Primary antibody for IdU: mouse anti‐BrdU (Becton Dickinson)
  • Secondary antibody for IdU: Texas Red–conjugated donkey anti–mouse Ig (Jackson ImmunoResearch)
  • High‐salt buffer (see recipe)
  • Vectashield mounting medium (Vector Labs)
  • Clear nail polish
  • 12‐mm‐diameter round glass coverslips, sterilized by flaming after dipping in 70% ethanol or by autoclaving
  • 100‐mm petri dishes and 24‐well plates
  • Humidified chamber (e.g., covered Tupperware container with moistened paper towels on the bottom)
  • Glass microscope slides
  • Fluorescence microscope (unit 4.2) with appropriate color filters for fluorophores used, e.g., blue (DAPI), green (FITC, Alexa Fluor 488), and red (e.g., Texas Red, Alexa Fluor 594)
  • Additional reagents and equipment for fluorescence microscopy (unit 4.2)

Basic Protocol 4: Replication Labeling of DNA Fibers

  Materials
  • Adherent tissue culture cells and appropriate medium
  • 10 mg/ml bromodeoxyuridine (BrdU) stock (store indefinitely at −20°C)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Lysis buffer (see recipe)
  • Fixative: 3:1 (v/v) methanol/acetic acid (prepare fresh and store on ice)
  • Blocking buffer: 1% (w/v) BSA in PBST (0.5% v/v Tween 20 in PBS)
  • PBST: 0.5% (v/v) Tween 20 in PBS
  • Primary antibody solution: mouse monoclonal anti‐BrdU (Becton Dickinson), diluted 1:10 in blocking buffer (see above)
  • Secondary antibody solution: Alexa Fluor 594–conjugated goat anti–mouse IgG (Molecular Probes) diluted 1:500 in blocking buffer (see above)
  • Vectashield mounting medium (Vector Labs)
  • 100‐mm culture dishes
  • 15‐ml conical centrifuge tubes
  • Centrifuge
  • Glass microscope slides
  • Diamond pen (optional)
  • Coplin jars
  • Humidified chamber (e.g., covered Tupperware container with moistened paper towels on the bottom)
  • Fluorescence microscope (unit 4.2) with appropriate color filters for fluorophores used, e.g., blue (DAPI), green (FITC, Alexa Fluor 488), and red (e.g., Texas Red, Alexa Fluor 594)
  • Additional reagents and equipment for cell culture (including trypsinization and cell counting; unit 1.1) and fluorescence microscopy (unit 4.2).
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Figures

Videos

Literature Cited

Literature Cited
   Anglana, M., Apiou, F., Bensimon, A., and Debatisse, M. 2003. Dynamics of DNA replication in mammalian somatic cells: Nucleotide pool modulates origin choice and interorigin spacing. Cell 114:385‐394.
   Baserga, R. and Malamud, D. 1969. Autoradiography: Technique and Application. Harper & Row, New York.
   Berezney, R., Dubey, D.D., and Huberman, J.A. 2000. Heterogeneity of eukaryotic replicons, replicon clusters, and replication foci. Chromosoma 108:471‐484.
   Bornfleth, H., Edelmann, P., Zink, D., Cremer, T., and Cremer, C. 1999. Quantitative motion analysis of subchromosomal foci in living cells using four‐dimensional microscopy. Biophys. J. 77:2871‐2886.
   Cleaver, J. 1967. Thymidine Metabolism and Cell Kinetics. North Holland, Amsterdam.
   Dimitrova, D.S. and Gilbert, D.M. 1999. The spatial position and replication timing of chromosomal domains are both established in early G1 phase. Mol. Cell. 4:983‐993.
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