In vitro initiation of bacteriophage phi 29 and M2 DNA replication: genes required for formation of a complex between

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Following Cell-fate in E. coli After Infection by Phage Lambda
06:10

Following Cell-fate in E. coli After Infection by Phage Lambda

Published on: October 14, 2011

K Matsumoto, T Saito, H Hirokawa

    Molecular & General Genetics : MGG
    |January 1, 1983
    PubMed

    Insights

    Bacterial phage DNA replication initiation was studied using cell-free extracts. Phage DNA replication initiation involves terminal protein-dAMP complex formation, reflecting in vivo processes.

    Area of Science:

    • Molecular Biology
    • Virology
    • Biochemistry

    Background:

    • Bacterial viruses, such as phi 29 and M2, employ unique strategies for DNA replication initiation.
    • Terminal protein (TP) is crucial for initiating replication in certain phages by forming a covalent linkage with the 5' ends of the DNA.

    Purpose of the Study:

    • To investigate the in vitro formation of the terminal protein-dAMP complex.
    • To determine if this in vitro complex formation reflects the in vivo initiation of phage DNA replication.

    Main Methods:

    • Preparation of cell-free extracts from Bacillus subtilis infected with phi 29 or M2 phages.
    • Assay for the formation of [alpha-32P]-dAMP-terminal protein complexes using radiolabeled nucleotides and phage DNA.
    • Complementation assays using extracts from cells infected with various phage mutants.

    Main Results:

    • Cell-free extracts from phi 29 and M2 infected cells catalyzed the formation of TP-dAMP complexes.
    • Complex formation was dependent on intact phage DNA and specific phage gene products, as evidenced by mutant studies and complementation.
    • The size of the phi 29 complex differed from the M2 complex due to variations in terminal protein molecular weights.

    Conclusions:

    • In vitro TP-dAMP complex formation accurately reflects the in vivo initiation of phage DNA replication.
    • The product of gene 2 of phi 29 is likely the enzyme responsible for catalyzing this initiation complex formation.

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