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A DNA primase specified by I-like plasmids.

E Lanka, E Scherzinger, E Günther

    Proceedings of the National Academy of Sciences of the United States of America
    |August 1, 1979
    PubMed
    Summary
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    Researchers isolated an enzyme from Escherichia coli that initiates DNA synthesis on single-stranded DNA. This enzyme substitutes for key host proteins and may be involved in conjugational DNA transfer.

    Area of Science:

    • Molecular Biology
    • Enzymology
    • Microbial Genetics

    Background:

    • Single-stranded DNA (ssDNA) replication is crucial for various biological processes, including viral replication and bacterial conjugation.
    • Escherichia coli possesses complex protein machinery for DNA replication initiation.

    Purpose of the Study:

    • To isolate and characterize a novel enzyme from E. coli involved in initiating DNA synthesis.
    • To elucidate the enzyme's role in ssDNA replication and its potential involvement in conjugational DNA transfer.

    Main Methods:

    • Enzyme isolation and purification from E. coli strains harboring specific plasmids.
    • In vitro assays to assess DNA synthesis initiation on various ssDNA templates.
    • Analysis of enzyme composition using SDS-PAGE and glycerol gradient centrifugation.

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  • Assays in E. coli extracts deficient in specific DNA polymerases and replication proteins.

    • Main Results:

    • An enzyme was isolated that initiates DNA synthesis on circular ssDNA of phages phi X174, fd, and G4.
    • The enzyme functionally substitutes for E. coli dnaB, dnaC, dnaG proteins, and RNA polymerase in vitro.
    • Optimal activity requires all four ribonucleoside triphosphates (rNTPs), with ATP, CTP, and GTP being nearly sufficient.
    • The enzyme specifically cooperates with DNA polymerase III, as evidenced by synthesis in polymerase I and II deficient extracts but not in dnaZ mutants.
    • Purified enzyme consists of two polypeptides (140,000 and 180,000 Mr) with a sedimentation coefficient of 3.6 S.
    • Elevated enzyme activity was observed in E. coli strains with derepressed conjugational plasmids, suggesting a role in conjugational DNA synthesis.

    Conclusions:

    • A novel enzyme from E. coli initiates DNA synthesis on ssDNA templates, mimicking key host replication factors.
    • The enzyme's dependence on specific rNTPs and its interaction with DNA polymerase III provide insights into its mechanism.
    • The correlation between enzyme activity and derepressed conjugational plasmids strongly suggests its involvement in conjugational DNA transfer.