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Related Experiment Videos

Yeast 2-micrometer plasmid DNA replication in vitro: origin and direction

H Kojo, B D Greenberg, A Sugino

    Proceedings of the National Academy of Sciences of the United States of America
    |December 1, 1981
    PubMed
    Summary

    Yeast Saccharomyces cerevisiae extracts replicate 2-micrometer DNA in vitro, mimicking cell cycle control. This discovery aids in purifying yeast DNA replication proteins.

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    Area of Science:

    • Molecular Biology
    • Yeast Genetics
    • DNA Replication

    Background:

    • Most yeast strains contain extrachromosomal 2-micrometer DNA, essential for replication.
    • 2-micrometer DNA synthesis is regulated by the cell cycle, similar to nuclear DNA.

    Purpose of the Study:

    • To establish an in vitro system for studying 2-micrometer DNA replication in Saccharomyces cerevisiae.
    • To investigate the cell cycle control mechanisms governing 2-micrometer DNA synthesis.

    Main Methods:

    • Utilized soluble extracts from Saccharomyces cerevisiae.
    • Performed semiconservative replication assays using 2-micrometer DNA and chimeric plasmids.
    • Analyzed replication intermediates, including theta forms, via gel electrophoresis and microscopy.

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    Main Results:

    • The yeast extract replicated 2-micrometer DNA and chimeric plasmids bidirectionally and discontinuously.
    • Replication initiated within inverted repeats, producing theta-shaped intermediates.
    • Extracts from cdc8 mutants exhibited temperature-sensitive replication, validating the in vitro system's biological relevance.

    Conclusions:

    • Developed a functional in vitro system for yeast 2-micrometer DNA replication.
    • The system accurately reflects in vivo cell cycle control of plasmid replication.
    • This assay is valuable for purifying and characterizing key yeast DNA replication factors.