Characterisation of RuvAB-Holliday junction complexes by glycerol gradient sedimentation

Kevin Hiom, Stephen C. West (Lead / Corresponding author)

    Research output: Contribution to journalArticle

    7 Citations (Scopus)

    Abstract

    The Escherichia coli RuvA and RuvB proteins interact specifically with Holliday junctions to promote ATP-dependent branch migration during genetic recombination and DNA repair. In the work described here, glycerol gradient centrifugation was used to investigate the requirements for the formation of pre-branch migration complexes. Since gradient centrifugation provides a simple and gentle method to analyse relatively unstable protein-DNA complexes, we were able to detect RuvA- and RuvAB-Holliday junction complexes without the need for chemical fixation. Using 35S-labelled RuvA protein and 3H-labelled Holliday junctions, we show that RuvA acts as a helicase accessory factor that loads the RuvB helicase onto the Holliday junction by structure-specific interactions. The resulting complex contained both RuvA and RuvB, as detected by Western blotting using serum raised against RuvA and RuvB. The stoichiometry of binding was estimated to be approximately four RuvA tetramers per junction. Formation of the RuvAB-Holliday junction complex required the presence of divalent metal ions and occurred without the need for ATP. However, the stability of the complex was enhanced by the presence of ATP gamma S, a non-hydrolysable ATP analogue. The data support a model for branch migration in which structure-specific binding of Holliday junctions by RuvA targets the assembly of hexameric RuvB rings on DNA. Specific loading of the RuvB ring helicase by RuvA is likely to be the initial step towards ATP-dependent branch migration.

    Original languageEnglish
    Pages (from-to)3621-3626
    Number of pages6
    JournalNucleic Acids Research
    Volume23
    Issue number18
    DOIs
    Publication statusPublished - 25 Sep 1995

    Fingerprint

    Cruciform DNA
    Glycerol
    Adenosine Triphosphate
    Centrifugation
    Recombinational DNA Repair
    Proteins
    DNA
    DNA Repair
    Genetic Recombination
    Western Blotting
    Metals
    Ions
    Escherichia coli
    Serum

    Keywords

    • Adenosine triphosphate
    • Bacterial proteins
    • Centrifugation, Density gradient
    • DNA
    • DNA helicases
    • DNA-binding proteins
    • Escherichia coli
    • Escherichia coli proteins
    • Glycerol
    • Magnesium
    • Oligodeoxyribonucleotides
    • Recombination, Genetic

    Cite this

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    title = "Characterisation of RuvAB-Holliday junction complexes by glycerol gradient sedimentation",
    abstract = "The Escherichia coli RuvA and RuvB proteins interact specifically with Holliday junctions to promote ATP-dependent branch migration during genetic recombination and DNA repair. In the work described here, glycerol gradient centrifugation was used to investigate the requirements for the formation of pre-branch migration complexes. Since gradient centrifugation provides a simple and gentle method to analyse relatively unstable protein-DNA complexes, we were able to detect RuvA- and RuvAB-Holliday junction complexes without the need for chemical fixation. Using 35S-labelled RuvA protein and 3H-labelled Holliday junctions, we show that RuvA acts as a helicase accessory factor that loads the RuvB helicase onto the Holliday junction by structure-specific interactions. The resulting complex contained both RuvA and RuvB, as detected by Western blotting using serum raised against RuvA and RuvB. The stoichiometry of binding was estimated to be approximately four RuvA tetramers per junction. Formation of the RuvAB-Holliday junction complex required the presence of divalent metal ions and occurred without the need for ATP. However, the stability of the complex was enhanced by the presence of ATP gamma S, a non-hydrolysable ATP analogue. The data support a model for branch migration in which structure-specific binding of Holliday junctions by RuvA targets the assembly of hexameric RuvB rings on DNA. Specific loading of the RuvB ring helicase by RuvA is likely to be the initial step towards ATP-dependent branch migration.",
    keywords = "Adenosine triphosphate, Bacterial proteins, Centrifugation, Density gradient, DNA, DNA helicases, DNA-binding proteins, Escherichia coli, Escherichia coli proteins, Glycerol, Magnesium, Oligodeoxyribonucleotides, Recombination, Genetic",
    author = "Kevin Hiom and West, {Stephen C.}",
    year = "1995",
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    doi = "10.1093/nar/23.18.3621",
    language = "English",
    volume = "23",
    pages = "3621--3626",
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    issn = "0305-1048",
    publisher = "Oxford University Press",
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    }

    Characterisation of RuvAB-Holliday junction complexes by glycerol gradient sedimentation. / Hiom, Kevin; West, Stephen C. (Lead / Corresponding author).

    In: Nucleic Acids Research, Vol. 23, No. 18, 25.09.1995, p. 3621-3626.

    Research output: Contribution to journalArticle

    TY - JOUR

    T1 - Characterisation of RuvAB-Holliday junction complexes by glycerol gradient sedimentation

    AU - Hiom, Kevin

    AU - West, Stephen C.

    PY - 1995/9/25

    Y1 - 1995/9/25

    N2 - The Escherichia coli RuvA and RuvB proteins interact specifically with Holliday junctions to promote ATP-dependent branch migration during genetic recombination and DNA repair. In the work described here, glycerol gradient centrifugation was used to investigate the requirements for the formation of pre-branch migration complexes. Since gradient centrifugation provides a simple and gentle method to analyse relatively unstable protein-DNA complexes, we were able to detect RuvA- and RuvAB-Holliday junction complexes without the need for chemical fixation. Using 35S-labelled RuvA protein and 3H-labelled Holliday junctions, we show that RuvA acts as a helicase accessory factor that loads the RuvB helicase onto the Holliday junction by structure-specific interactions. The resulting complex contained both RuvA and RuvB, as detected by Western blotting using serum raised against RuvA and RuvB. The stoichiometry of binding was estimated to be approximately four RuvA tetramers per junction. Formation of the RuvAB-Holliday junction complex required the presence of divalent metal ions and occurred without the need for ATP. However, the stability of the complex was enhanced by the presence of ATP gamma S, a non-hydrolysable ATP analogue. The data support a model for branch migration in which structure-specific binding of Holliday junctions by RuvA targets the assembly of hexameric RuvB rings on DNA. Specific loading of the RuvB ring helicase by RuvA is likely to be the initial step towards ATP-dependent branch migration.

    AB - The Escherichia coli RuvA and RuvB proteins interact specifically with Holliday junctions to promote ATP-dependent branch migration during genetic recombination and DNA repair. In the work described here, glycerol gradient centrifugation was used to investigate the requirements for the formation of pre-branch migration complexes. Since gradient centrifugation provides a simple and gentle method to analyse relatively unstable protein-DNA complexes, we were able to detect RuvA- and RuvAB-Holliday junction complexes without the need for chemical fixation. Using 35S-labelled RuvA protein and 3H-labelled Holliday junctions, we show that RuvA acts as a helicase accessory factor that loads the RuvB helicase onto the Holliday junction by structure-specific interactions. The resulting complex contained both RuvA and RuvB, as detected by Western blotting using serum raised against RuvA and RuvB. The stoichiometry of binding was estimated to be approximately four RuvA tetramers per junction. Formation of the RuvAB-Holliday junction complex required the presence of divalent metal ions and occurred without the need for ATP. However, the stability of the complex was enhanced by the presence of ATP gamma S, a non-hydrolysable ATP analogue. The data support a model for branch migration in which structure-specific binding of Holliday junctions by RuvA targets the assembly of hexameric RuvB rings on DNA. Specific loading of the RuvB ring helicase by RuvA is likely to be the initial step towards ATP-dependent branch migration.

    KW - Adenosine triphosphate

    KW - Bacterial proteins

    KW - Centrifugation, Density gradient

    KW - DNA

    KW - DNA helicases

    KW - DNA-binding proteins

    KW - Escherichia coli

    KW - Escherichia coli proteins

    KW - Glycerol

    KW - Magnesium

    KW - Oligodeoxyribonucleotides

    KW - Recombination, Genetic

    U2 - 10.1093/nar/23.18.3621

    DO - 10.1093/nar/23.18.3621

    M3 - Article

    C2 - 7478987

    VL - 23

    SP - 3621

    EP - 3626

    JO - Nucleic Acids Research

    JF - Nucleic Acids Research

    SN - 0305-1048

    IS - 18

    ER -