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Related Experiment Videos

CDC7-dependent protein kinase activity in yeast replicative-complex preparations.

S M Jazwinski1

  • 1Department of Biochemistry and Molecular Biology, Louisiana State University Medical Center, New Orleans 70112.

Proceedings of the National Academy of Sciences of the United States of America
|April 1, 1988
PubMed
Summary

A novel protein kinase activity in yeast DNA replication complexes is dependent on the CDC7 gene. This suggests a role for phosphorylation in controlling DNA replication initiation during the cell cycle.

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Area of Science:

  • Molecular Biology
  • Cell Cycle Regulation
  • Yeast Genetics

Background:

  • DNA replication is a fundamental process tightly regulated during the cell cycle.
  • Protein phosphorylation plays a critical role in cell cycle control.
  • The budding yeast Saccharomyces cerevisiae is a model organism for studying DNA replication and cell cycle progression.

Purpose of the Study:

  • To identify and characterize protein kinase activity associated with the DNA replicative complex in Saccharomyces cerevisiae.
  • To investigate the relationship between this protein kinase activity and the cell division cycle 7 (CDC7) gene product.
  • To explore the potential role of this kinase activity in the regulation of DNA replication initiation.

Main Methods:

  • Isolation and preparation of DNA-replicative complexes from Saccharomyces cerevisiae.

Related Experiment Videos

  • Assay of endogenous protein kinase activity and substrate specificity.
  • Characterization of kinase activity using various activators and divalent cations.
  • Analysis of kinase activity in cell division cycle 7 (cdc7) mutant strains at restrictive temperatures.
  • Copurification studies of protein kinase and DNA replicating activities.

    • Main Results:

    • A protein kinase activity was identified in yeast DNA-replicative complexes, preferentially phosphorylating a 48-kDa polypeptide on serine residues.
    • The kinase activity was not stimulated by common second messengers (cAMP, cGMP, Ca2+) and showed a specific requirement for Mg2+.
    • The protein kinase activity was heat-sensitive in replicative fractions from cdc7 mutants, indicating CDC7-dependence.
    • The kinase activity copurified with DNA replicating activity, suggesting an interaction with the yeast replicative complex.

    Conclusions:

    • The CDC7 gene product is involved in regulating DNA replication initiation in yeast.
    • The identified protein kinase activity, potentially linked to CDC7, may play a role in controlling the initiation of DNA replication.
    • Phosphorylation of replication machinery components could be a mechanism for regulating cell cycle progression at the G1/S boundary.