Assays for CDK Activity and DNA Replication in the Cell Cycle

Jonathon Pines1, Mark Jackman1, Karen Simpson1

1 Wellcome/CRC Institute, and Cambridge University, Cambridge, United Kingdom
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 8.2
DOI:  10.1002/0471143030.cb0802s00
Online Posting Date:  May, 2001
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Abstract

This unit describes two assays for different stages of the cell cycle in tissue culture cells. One is a biochemical measurement that assays the protein kinase activity of different cyclin‚Äźdependent kinase complexes that are present in late G1 phase, S phase, G2 phase, or mitosis. The other assay uses immunofluorescence to detect DNA replication by the incorporation of nucleotide analogs into DNA. These assays are useful in analyzing the stage and degree of synchrony of the cell cycle and changes in the basic cell cycle machinery.

     
 
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Table of Contents

  • Basic Protocol 1: Measuring CDK Activity
  • Basic Protocol 2: Measuring DNA Replication Using Incorporation of BrdU
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Measuring CDK Activity

  Materials
  • Cells grown in 6‐cm tissue culture dishes
  • 1× PBS ( appendix 2A), ice cold
  • Lysis buffer (see recipe), ice cold
  • Protein A– or protein G–Sepharose conjugate (Amersham Pharmacia Biotech) or formaldehyde‐treated Staphylococcus aureus cells (Pansorbin, Calbiochem)
  • Anti‐cyclin antibody, anti‐CDK antibody, or Cks–Sepharose bead conjugate (Upstate Biotechnology)
  • Kinase buffer (see recipe), ice cold
  • Histone H1 solution (see recipe) or consensus cdc2 peptide (New England Biolabs)
  • 1 mM ATP (diluted from 100 mM ATP [see recipe] in distilled H 2O)
  • 2000 Ci/mmol [γ‐33P]ATP or 3000 Ci/mmol [γ‐32P]ATP
  • 100 mM EDTA ( appendix 2A)
  • 2× SDS sample buffer (unit 6.1)
  • PKA‐inhibitory peptide: 1 mM of peptide (Sigma) in 10 mM sodium phosphate, pH 7.2 (see appendix 2A for buffer recipe; store peptide solution up to 1 year at –20°C)
  • 50 mM roscovitine or 100 mM olomoucine (both from Calbiochem) in DMSO (store both solutions in aliquots up to 1 year at –20°C, and thaw only once; store olomoucine in the dark)
  • Negative control: immunoprecipitated preimmune serum or anti‐IgG antibody
  • Positive control: purified cyclin B–CDK1
  • 75 mM phosphoric acid (7.5 ml of 1 M phosphoric acid + 92.5 ml distilled water; store up to 2 years at room temperature)
  • 96% ethanol
  • 15% to 20% SDS‐polyacrylamide gel (unit 6.1)
  • Coomassie blue G‐250 staining solution (see unit 6.1)
  • Destaining solution: 10% (v/v) acetic acid
  • 1‐ml syringes and 21‐G needles, prechilled to 4°C
  • 1.5‐ml microcentrifuge tubes, prechilled to 4°C
  • 1‐ml pipet tips, prechilled to 4°C
  • Phosphocellulose units (Pierce) or 1.5‐cm squares of phosphocellulose P81 filter paper (Whatman)
  • Microcentrifuge, 4°C
  • End‐over‐end rotator or rotating wheel
  • Whatman 3MM filter paper
  • Phosphorimager (optional)
  • Additional reagents and equipment for SDS‐PAGE and Coomassie brilliant blue staining (unit 6.1) and for autoradiography and densitometry (optional; unit 6.3)

Basic Protocol 2: Measuring DNA Replication Using Incorporation of BrdU

  Materials
  • 1 mg/ml poly‐L‐lysine: 20 mg poly‐L‐lysine (average mol. wt. 400,000)/20 ml dH 2O; filter sterilize through 0.45 µm filter and store at –20°C for 3 months
  • Cell culture medium
  • Cell suspension: 105 cells/ml in PBS (unit 1.1; see appendix 2A for PBS recipe)
  • 10 mg/ml BrdU (see recipe)
  • 1 mg/ml 5‐fluorodeoxyuridine (FrdU; see recipe)
  • PBS ( appendix 2A)
  • Blocking solution: 3% (w/v) BSA in PBS
  • 50% methanol/50% acetone (fixative; prepared fresh); or formaldehyde solution (cross‐linking agent; see recipe) and Tris⋅Cl/MgCl 2/Triton X‐100(TSM; see recipe)
  • EcoR1 exonuclease and exonuclease III, or HCl/Triton X‐100 (see recipe)
  • Anti‐BrdU antibody, conjugated; or unconjugated anti‐BrdU antibody and fluorophore‐labeled secondary antibody (Amersham)
  • Slide‐mounting solution containing antifade agent
  • Grade 1 (0.15‐mm) glass coverslips, cleaned and sterilized with dry heat
  • Cytocentrifuge (optional)
  • Humidified chamber: e.g., inverted petri dish with dampened filter paper in lid
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Figures

Videos

Literature Cited

Literature Cited
   Harlow, E. and Lane, D.P. 1988. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
   Jeffrey, P.D., Russo, A.A., Polyak, K., Gibbs, E., Hurwitz, J., Massague, J., and Pavletich, N.P. 1995. Structure of a cyclin A–CDK2 complex. Nature 376:313‐320.
   Lew, D.J. and Kornbluth, S. 1996. Regulatory roles of cyclin‐dependent kinase phosphorylation in cell cycle control. Curr. Opin. Cell. Biol. 8:795‐804.
   Matsushime, H., Roussel, M.F., Ashmun, R.A., and Sherr, C.J. 1991. Colony‐stimulating factor 1 regulates novel cyclins during the G1 phase of the cell cycle. Cell 65:701‐713.
   Morgan, D.O. 1995. Principles of CDK regulation. Nature 374:131‐134.
   Phelps, D.E. and Xiong, Y. 1997. Assay for activity of mammalian cyclin D–dependent kinases CDK4 and CDK6. Methods Enzymol. 283:194‐205.
   Yamashita, K., Yasuda, H., Pines, J., Yasumoto, K., Nishitani, H., Ohtsubo, M., Hunter, T., Sugimura, T., and Nishimoto, T. 1991. Okadaic acid, a potent inhibitor of type 1 and type 2A protein phosphatases, activates cdc2/H1 kinase and transiently induces a premature mitosis‐like state in BHK21 cells. EMBO J. 9:4331‐4338.
Key Reference
   Dunphy, W.G. ed. 1997. Methods Enzymol. Vol. 283: Cell Cycle Control. Academic Press New York.
  Presents a wide variety of protocols for analyzing the cell cycle.
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