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Related Experiment Videos

Formation of rolling-circle molecules during phi X174 complementary strand DNA replication.

M Mok, K J Marians

    The Journal of Biological Chemistry
    |February 15, 1987
    PubMed
    Summary

    The Escherichia coli primosome, a protein complex, functions as a DNA helicase. This unwinding activity is crucial for the rolling circle DNA replication mechanism observed in coliphage phi X174.

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    Area of Science:

    • Molecular Biology
    • Virology
    • Biochemistry

    Background:

    • The primosome is a complex protein apparatus essential for DNA replication initiation in Escherichia coli.
    • Its role as a DNA helicase, in addition to its DNA G-catalyzed priming function, has been debated.
    • The primosome comprises seven E. coli proteins, including products of dnaB, dnaC, dnaG genes, replication factor Y, and proteins i, n, and n'.

    Purpose of the Study:

    • To investigate the primosome's role in unwinding duplex DNA strands during DNA replication.
    • To determine if the primosome possesses DNA helicase activity.
    • To elucidate the primosome's contribution to the rolling circle replication mechanism.

    Main Methods:

    • In vitro DNA replication assay using coliphage phi X174 SS(c) replicative form DNA.

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  • Requirement analysis of E. coli single-stranded DNA binding protein, primosomal proteins, and DNA polymerase III holoenzyme.
  • Comparison of DNA replication with and without primosome assembly sites, and with a general priming system.

    • Main Results:

    • Multigenome-length, linear, double-stranded DNA molecules were generated, indicating a rolling circle mechanism.
    • Helicase-catalyzed strand-displacement activity was necessary for continuous complementary strand synthesis.
    • Templates lacking primosome assembly sites failed to support linear multimer production, and a general priming system showed reduced linear multimer synthesis.

    Conclusions:

    • The primosome actively participates in DNA helicase activity, unwinding duplex DNA strands.
    • This helicase function is essential for the efficient rolling circle replication of coliphage phi X174 DNA.
    • The primosome's specific assembly sites are critical for its helicase function in this replication process.