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Bacteriophage Mu DNA replication in vitro.

N P Higgins, D Moncecchi, P Manlapaz-Ramos

    The Journal of Biological Chemistry
    |April 10, 1983
    PubMed
    Summary
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    Bacteriophage Mu DNA replication in vitro is semidiscontinuous, with Okazaki fragments primarily on the light strand. Host DNA sequences are integrated into Mu viral DNA, suggesting recruitment of bacterial replication machinery.

    Area of Science:

    • Molecular Biology
    • Virology
    • Genetics

    Background:

    • Bacteriophage Mu is a versatile genetic element with complex replication mechanisms.
    • Understanding phage DNA replication is crucial for gene transfer and manipulation studies.

    Purpose of the Study:

    • To establish and utilize an in vitro system for studying bacteriophage Mu DNA replication.
    • To elucidate the mechanism and strand bias of Mu DNA synthesis and Okazaki fragment formation.

    Main Methods:

    • Development of an in vitro replication system using cell lysates on cellophane discs.
    • Monitoring Mu replication via DNA hybridization techniques.
    • Utilizing a thermoinducible Mu lysogen for specific Mu DNA synthesis analysis.
    • Employing a DNA ligase inhibitor (NMN) to distinguish Okazaki pieces from larger DNA fragments.

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    Main Results:

    • 30-50% of in vitro DNA synthesis was Mu-specific in a thermoinducible lysogen.
    • Mu DNA replication was found to be semidiscontinuous.
    • Mu Okazaki pieces predominantly hybridized to the Mu light strand, while larger DNA fragments hybridized to the heavy strand.
    • Host DNA sequences were found in mature Mu viral DNA, with Okazaki pieces hybridizing to both Mu strands.

    Conclusions:

    • The in vitro system effectively recapitulates key aspects of bacteriophage Mu DNA replication.
    • Mu DNA replication exhibits strand asymmetry, with Okazaki pieces associated with the light strand.
    • Bacterial replication machinery is likely recruited by Mu, traversing the genome from left to right, incorporating host sequences.