Proteolysis and DNA replication: The CDC34 requirement in the Xenopus egg cell cycle

Science

Volumn 277, Issue 5332, 1997, Pages 1672-1676

  Yew, P Renee ^a   Kirschner, Marc W ^a  

^a HARVARD MEDICAL SCHOOL   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

CELL CYCLE PROTEIN; CYCLIN DEPENDENT KINASE; CYCLIN E; GENE PRODUCT; UBIQUITIN;

ANIMAL CELL; ARTICLE; CELL CYCLE; CONTROLLED STUDY; DNA REPLICATION; EMBRYO; EMBRYO DEVELOPMENT; NONHUMAN; PRIORITY JOURNAL; PROTEIN DEGRADATION; XENOPUS;

ANIMALS; CDC2-CDC28 KINASES; CELL CYCLE PROTEINS; CELL NUCLEUS; CHROMOSOMES; CYCLIN-DEPENDENT KINASE 2; CYCLIN-DEPENDENT KINASE INHIBITOR P27; CYCLIN-DEPENDENT KINASES; CYCLINS; DNA REPLICATION; ENZYME INHIBITORS; HUMANS; LIGASES; MICROTUBULE-ASSOCIATED PROTEINS; MITOSIS; MUTATION; OVUM; PROTEIN-SERINE-THREONINE KINASES; PROTEINS; RECOMBINANT PROTEINS; S PHASE; TUMOR SUPPRESSOR PROTEINS; UBIQUITIN-PROTEIN LIGASE COMPLEXES; UBIQUITIN-PROTEIN LIGASES; XENOPUS LAEVIS; XENOPUS PROTEINS;

ANIMALIA; EUKARYOTA; SACCHAROMYCETALES; XENOPUS LAEVIS;

EID: 0030812809     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.277.5332.1672     Document Type: Article

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27. LSS was generated as described (16). Extracts replicated 75 to 100% of the input template (5 ng/μl). Cycloheximide (0.1 mg/ml) was added to LSS before replication was assayed.
28. Demembranated sperm nuclei were prepared as described (4) and used at a final DNA concentration of 5 ng/μl. Plasmid template ssM13 DNA was added to a 10 ng/μl final concentration. DNA replication was measured by trichloroacetic acid precipitation of radiolabeled fragments (4, 10) and presented as a percent replication of the input template (5 ng/μl). Alternatively, samples were ethanol precipitated, run on agarose gels, and autoradiographed.
29. Antibodies were covalently cross-linked to protein A (pA)-Sepharose (Seph) or pA-affiprep beads as described [E. Harlow and D. Lane, Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1988)]. Immunodepletion was performed by mixing 1 volume LSS to 1.2 volumes antibody-coupled pA-Seph beads for 2 hours at 0°C with resuspension every 10 min.
30. LSS, Superdex column fraction, or eluted affiprep material were analyzed by SDS-PAGE, transferred to nitrocellulose, incubated with affinity-purified anti-hCDC34 immunoglobulin (Ig), and analyzed by enhanced chemiluminescence (Amersham).
31. Replication foci were analyzed by stated methods (10, 12).
32. 2, and 10 mM Hepes (pH 7.7)] (one time), XB-with 500 mM KCl (three times), and 0.5× XB-(two times) (23). Immunodepleted LSS was added to the beads and assayed for replication. Alternatively, the washed beads were treated with 100mM glycine (pH 2.5), and the eluate was neutralized with 1 M tris (pH 8). Samples were concentrated and analyzed by SDS-PAGE and silver stained or analyzed by immunoblotting.
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35. 35S]methionine. Xic1 bands were quantitated by phosphorimager (Molecular Dynamics), and Xic1 levels were normalized to the percent Xic1 remaining to determine half-life values.
36. 35S]met-labeled Xic1 was used, and sperm nuclei were added to a concentration of 3.2 ng/μl (1000 nuclei/μl), 7.9 ng/μl (2500 nuclei/ μl), or 15.8 ng/μl (5000 nuclei/μl) and incubated for 3 hours at 23°C.
37. We thank S. Plon for hCDC34/pGEX; J. Maller for baculovirus Cdk2-cyclin E, Cdk2, kinase-inactive Cdk2, and Xic1/pBluescript; A. Philpott for sperm nuclei and HSS; R. King for Δ90; V. Chau for sharing results before publication; J. Ruderman, D. Finley, A. Philpott, T. Boyer, R. King, P. Adams, and R. Davis for critical reading of the manuscript; A. Philpott, J. Peters, T. Boyer, R. King, and P. Jackson for helpful discussions; and the National Institutes of General Medical Science for support of this work. P.R.Y. is a fellow of the Jane Coffin Childs Memorial Fund.