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Related Experiment Videos

A DNA primase from yeast. Purification and partial characterization.

S M Jazwinski, G M Edelman

    The Journal of Biological Chemistry
    |April 25, 1985
    PubMed
    Summary

    Researchers purified a nearly homogeneous DNA primase from yeast, finding it functions as a monomer and synthesizes short DNA primers. This enzyme requires ribonucleotides to start primer synthesis but can incorporate deoxynucleotides, with some activity associating with DNA polymerase.

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    Area of Science:

    • Molecular Biology
    • Biochemistry
    • Yeast Genetics

    Background:

    • DNA primase is essential for initiating DNA replication by synthesizing RNA primers.
    • Understanding DNA primase function in yeast provides insights into eukaryotic DNA replication mechanisms.

    Purpose of the Study:

    • To purify and characterize DNA primase activity from Saccharomyces cerevisiae.
    • To investigate the enzymatic properties and potential associations of yeast DNA primase.

    Main Methods:

    • Purification of DNA primase using chromatography and sedimentation techniques.
    • Enzymatic assays to determine template usage, cofactor requirements, and product synthesis.
    • Molecular weight determination via sedimentation and chromatography.

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    Main Results:

    • A nearly homogeneous DNA primase preparation was obtained, devoid of DNA and RNA polymerase activities.
    • The primase activity was associated with a 65 kDa polypeptide, suggesting a monomeric enzyme.
    • The enzyme utilized denatured DNA and poly(dT) templates, requiring divalent cations and rATP for initiation, and could incorporate deoxynucleotides into primers.
    • Primase synthesized oligonucleotides of 11-13 nucleotides, with some association observed between primase and DNA polymerase activities.

    Conclusions:

    • The purified yeast DNA primase is a distinct enzymatic entity with specific substrate and cofactor requirements.
    • The enzyme's monomeric nature and its association with DNA polymerase suggest conserved mechanisms in eukaryotic DNA replication.