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Initiation of genetic exchanges in lambda phage--prophage crosses.

P F Lin, E Bardwell, P Howard-Flanders

    Proceedings of the National Academy of Sciences of the United States of America
    |January 1, 1977
    PubMed
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    Psoralen damage to lambda phage DNA significantly increases genetic exchange frequencies in Escherichia coli lysogens. This enhanced recombination requires DNA repair enzymes, particularly UV-endonuclease and RecA, suggesting crosslinks initiate the process.

    Area of Science:

    • Molecular Biology
    • Genetics
    • Microbiology

    Background:

    • Bacteriophage lambda (λ) infection of Escherichia coli K-12 lysogens normally results in low-frequency genetic exchange between phage and prophage.
    • Photosensitizing agents like 4,5',8-trimethylpsoralen, when combined with UV light, can induce DNA damage.

    Purpose of the Study:

    • To investigate the effect of psoralen-induced DNA damage on the frequency of genetic exchange between infecting lambda phages and prophages in E. coli.
    • To elucidate the role of host cell DNA repair and recombination pathways in mediating psoralen-induced genetic exchanges.

    Main Methods:

    • Infection of various Escherichia coli K-12 lysogenic strains (including wild-type and mutants deficient in DNA repair genes like recB, recC, recF, lexA, uvrA, uvrB, and recA) with lambda phages.

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  • Treatment of infecting lambda phages with 4,5',8-trimethylpsoralen and 360 nm light prior to infection.
  • Measurement of genetic exchange frequencies between phage and prophage.
  • Analysis of DNA crosslink formation using alkaline sedimentation and kinetic studies.

    • Main Results:

    • Psoralen treatment and light significantly increased genetic exchange frequencies (above 1%) compared to untreated phages (less than 0.1%).
    • Psoralen-induced exchanges were observed in wild-type and recB, recC, recF, and lexA mutant lysogens, but were significantly reduced in uvrA, uvrB, and recA mutant strains.
    • DNA crosslink formation showed second-order kinetics, suggesting a two-photon reaction, and correlated with the observed increase in genetic exchanges.

    Conclusions:

    • Psoralen-induced DNA crosslinks in phage DNA, rather than monoadducts, are likely responsible for the elevated genetic exchange frequencies.
    • The host cell's DNA repair machinery, specifically involving UV-endonuclease (UvrA) and RecA protein, is crucial for processing psoralen-induced crosslinks and initiating recombination.
    • Recombination steps following the initial processing of crosslinks, including homologous pairing, cutting, and joining, require the recA(+) gene product.