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Assays for CDK Activity and DNA Replication in the Cell Cycle

Jonathon Pines¹, Mark Jackman¹, Karen Simpson¹

¹Wellcome/CRC Institute, and Cambridge University, Cambridge, United Kingdom, 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-dependent 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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Unit Introduction
Basic Protocol 1: Measuring CDK Activity
Basic Protocol 2: Measuring DNA Replication Using Incorporation of BrdU
Reagents and Solutions
Commentary
Bibliography
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 H₂O)
2000 Ci/mmol [-³³P]ATP or 3000 Ci/mmol [-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 dH₂O; 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/MgCl₂/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

Figure 8.2.1

Appearance and disappearance of specific cyclin-cell-dependent kinase (CDK) activities through the cell cycle. The arrow marks the end of G₂ phase, at which point cyclin B–CDK1 is activated by the Cdc25 phosphatase. The relative kinase activities are not to scale. Abbreviations: M, metaphase; A, anaphase.

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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.