Alleviation of EcoK DNA restriction in Escherichia coli and involvement of umuDC activity

Kevin J. Hiom, Steven G. Sedgwick

    Research output: Contribution to journalArticle

    18 Citations (Scopus)

    Abstract

    The activity of the EcoK DNA restriction system of Escherichia coli reduces both the plating efficiency of unmodified phage lambda and the transforming ability of unmodified pBR322 plasmid DNA. However, restriction can be alleviated in wild-type cells, by UV irradiation and expression of the SOS response, so that 10(3)- to 10(4)-fold increases in phage growth and fourfold increases in plasmid transformation occurred with unmodified DNA. Restriction alleviation was found to be a transient effect because induced cells, which initially failed to restrict unmodified plasmid DNA, later restricted unmodified phage lambda. Although the SOS response was needed for restriction alleviation, constitutive SOS induction, elicited genetically with a recA730 mutation, did not alleviate restriction and UV irradiation was still needed. A hitherto unsuspected involvement of the umuDC operon in this alleviation of restriction is characterized and, by differential complementation, was separated from the better known role of umuDC in mutagenic DNA repair. The need for cleavage of UmuD for restriction alleviation was shown with plasmids encoding cleavable, cleaved, and non-cleavable forms of UmuD. However, UV irradiation was still needed even when cleaved UmuD was provided. The possibility that restriction alleviation occurs by a general inhibition of the EcoK restriction/modification complex was tested and discounted because modification of lambda was not reduced by UV irradiation. An alternative idea, that restriction activity was competitively reduced by an increase in EcoK modification, was also discounted by the lack of any increase in the modification of lambda Ral-, a naturally undermodified phage. Other possible mechanisms for restriction alleviation are discussed.

    Original languageEnglish
    Pages (from-to)265-275
    Number of pages11
    JournalMolecular & General Genetics : MGG
    Volume231
    Issue number2
    DOIs
    Publication statusPublished - Jan 1992

    Fingerprint

    Plasmids
    Escherichia coli
    Bacteriophage lambda
    DNA
    Bacteriophages
    Operon
    DNA Repair
    Mutation
    Growth

    Keywords

    • Bacterial Proteins
    • Bacteriophage lambda
    • DNA Damage
    • DNA Transposable Elements
    • DNA, Bacterial
    • DNA-Directed DNA Polymerase
    • Deoxyribonucleases, Type I Site-Specific
    • Escherichia coli
    • Escherichia coli Proteins
    • Genes, Bacterial
    • Genetic Complementation Test
    • Hydrolysis
    • Mutagenesis
    • Operon
    • Plasmids
    • SOS Response (Genetics)
    • Ultraviolet Rays

    Cite this

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    title = "Alleviation of EcoK DNA restriction in Escherichia coli and involvement of umuDC activity",
    abstract = "The activity of the EcoK DNA restriction system of Escherichia coli reduces both the plating efficiency of unmodified phage lambda and the transforming ability of unmodified pBR322 plasmid DNA. However, restriction can be alleviated in wild-type cells, by UV irradiation and expression of the SOS response, so that 10(3)- to 10(4)-fold increases in phage growth and fourfold increases in plasmid transformation occurred with unmodified DNA. Restriction alleviation was found to be a transient effect because induced cells, which initially failed to restrict unmodified plasmid DNA, later restricted unmodified phage lambda. Although the SOS response was needed for restriction alleviation, constitutive SOS induction, elicited genetically with a recA730 mutation, did not alleviate restriction and UV irradiation was still needed. A hitherto unsuspected involvement of the umuDC operon in this alleviation of restriction is characterized and, by differential complementation, was separated from the better known role of umuDC in mutagenic DNA repair. The need for cleavage of UmuD for restriction alleviation was shown with plasmids encoding cleavable, cleaved, and non-cleavable forms of UmuD. However, UV irradiation was still needed even when cleaved UmuD was provided. The possibility that restriction alleviation occurs by a general inhibition of the EcoK restriction/modification complex was tested and discounted because modification of lambda was not reduced by UV irradiation. An alternative idea, that restriction activity was competitively reduced by an increase in EcoK modification, was also discounted by the lack of any increase in the modification of lambda Ral-, a naturally undermodified phage. Other possible mechanisms for restriction alleviation are discussed.",
    keywords = "Bacterial Proteins, Bacteriophage lambda, DNA Damage, DNA Transposable Elements, DNA, Bacterial, DNA-Directed DNA Polymerase, Deoxyribonucleases, Type I Site-Specific, Escherichia coli, Escherichia coli Proteins, Genes, Bacterial, Genetic Complementation Test, Hydrolysis, Mutagenesis, Operon, Plasmids, SOS Response (Genetics), Ultraviolet Rays",
    author = "Hiom, {Kevin J.} and Sedgwick, {Steven G.}",
    year = "1992",
    month = "1",
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    language = "English",
    volume = "231",
    pages = "265--275",
    journal = "Molecular & General Genetics : MGG",
    issn = "0026-8925",
    publisher = "Springer Verlag",
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    }

    Alleviation of EcoK DNA restriction in Escherichia coli and involvement of umuDC activity. / Hiom, Kevin J.; Sedgwick, Steven G.

    In: Molecular & General Genetics : MGG, Vol. 231, No. 2, 01.1992, p. 265-275.

    Research output: Contribution to journalArticle

    TY - JOUR

    T1 - Alleviation of EcoK DNA restriction in Escherichia coli and involvement of umuDC activity

    AU - Hiom, Kevin J.

    AU - Sedgwick, Steven G.

    PY - 1992/1

    Y1 - 1992/1

    N2 - The activity of the EcoK DNA restriction system of Escherichia coli reduces both the plating efficiency of unmodified phage lambda and the transforming ability of unmodified pBR322 plasmid DNA. However, restriction can be alleviated in wild-type cells, by UV irradiation and expression of the SOS response, so that 10(3)- to 10(4)-fold increases in phage growth and fourfold increases in plasmid transformation occurred with unmodified DNA. Restriction alleviation was found to be a transient effect because induced cells, which initially failed to restrict unmodified plasmid DNA, later restricted unmodified phage lambda. Although the SOS response was needed for restriction alleviation, constitutive SOS induction, elicited genetically with a recA730 mutation, did not alleviate restriction and UV irradiation was still needed. A hitherto unsuspected involvement of the umuDC operon in this alleviation of restriction is characterized and, by differential complementation, was separated from the better known role of umuDC in mutagenic DNA repair. The need for cleavage of UmuD for restriction alleviation was shown with plasmids encoding cleavable, cleaved, and non-cleavable forms of UmuD. However, UV irradiation was still needed even when cleaved UmuD was provided. The possibility that restriction alleviation occurs by a general inhibition of the EcoK restriction/modification complex was tested and discounted because modification of lambda was not reduced by UV irradiation. An alternative idea, that restriction activity was competitively reduced by an increase in EcoK modification, was also discounted by the lack of any increase in the modification of lambda Ral-, a naturally undermodified phage. Other possible mechanisms for restriction alleviation are discussed.

    AB - The activity of the EcoK DNA restriction system of Escherichia coli reduces both the plating efficiency of unmodified phage lambda and the transforming ability of unmodified pBR322 plasmid DNA. However, restriction can be alleviated in wild-type cells, by UV irradiation and expression of the SOS response, so that 10(3)- to 10(4)-fold increases in phage growth and fourfold increases in plasmid transformation occurred with unmodified DNA. Restriction alleviation was found to be a transient effect because induced cells, which initially failed to restrict unmodified plasmid DNA, later restricted unmodified phage lambda. Although the SOS response was needed for restriction alleviation, constitutive SOS induction, elicited genetically with a recA730 mutation, did not alleviate restriction and UV irradiation was still needed. A hitherto unsuspected involvement of the umuDC operon in this alleviation of restriction is characterized and, by differential complementation, was separated from the better known role of umuDC in mutagenic DNA repair. The need for cleavage of UmuD for restriction alleviation was shown with plasmids encoding cleavable, cleaved, and non-cleavable forms of UmuD. However, UV irradiation was still needed even when cleaved UmuD was provided. The possibility that restriction alleviation occurs by a general inhibition of the EcoK restriction/modification complex was tested and discounted because modification of lambda was not reduced by UV irradiation. An alternative idea, that restriction activity was competitively reduced by an increase in EcoK modification, was also discounted by the lack of any increase in the modification of lambda Ral-, a naturally undermodified phage. Other possible mechanisms for restriction alleviation are discussed.

    KW - Bacterial Proteins

    KW - Bacteriophage lambda

    KW - DNA Damage

    KW - DNA Transposable Elements

    KW - DNA, Bacterial

    KW - DNA-Directed DNA Polymerase

    KW - Deoxyribonucleases, Type I Site-Specific

    KW - Escherichia coli

    KW - Escherichia coli Proteins

    KW - Genes, Bacterial

    KW - Genetic Complementation Test

    KW - Hydrolysis

    KW - Mutagenesis

    KW - Operon

    KW - Plasmids

    KW - SOS Response (Genetics)

    KW - Ultraviolet Rays

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    U2 - 10.1007/BF00279800

    DO - 10.1007/BF00279800

    M3 - Article

    VL - 231

    SP - 265

    EP - 275

    JO - Molecular & General Genetics : MGG

    JF - Molecular & General Genetics : MGG

    SN - 0026-8925

    IS - 2

    ER -