Green fluorescent protein fused to the C terminus ofRAD51 specifically interferes with secondary DNAbinding by the RAD51-ssDNA complex

Wataru Kobayashi, Satoshi Sekine, Shinichi Machida, Hitoshi Kurumizaka

    Research output: Contribution to journalArticle

    6 Citations (Scopus)

    Abstract

    Green fluorescent protein (GFP), fused to the N or C terminus of a protein of interest, is widely used to monitor the localization and mobility of proteins in cells. RAD51 is an essential protein that functions in mitotic DNA repair and meiotic chromosome segregation by promoting the homologous recombination reaction. A previous genetic study with Arabidopsis thaliana revealed that GFP fused to the C terminus of RAD51 (RAD51-GFP) inhibits mitotic DNA repair, but meiotic homologous recombination remained unaffected. To determine how the C-terminal GFP specifically inhibits mitotic DNA repair by RAD51, we purified rice RAD51A1-GFP and RAD51A2-GFP, and performed biochemical analyses.Interestingly, purified RAD51A1-GFP and RAD51A2-GFP are proficient in DNA binding and ATP hydrolysis. However, nucleoprotein complexes containing single-stranded DNA and RAD51A1-GFP or RAD51A2-GFP are significantly defective in binding to the second DNA molecule (secondary DNA binding), and consequently fail to catalyze homologous pairing. In contrast, RAD51A1-GFP and RAD51A2-GFP efficiently stimulated homologous pairing promoted by the meiosis-specific RAD51 isoform DMC1. These biochemical characteristics are well conserved in human RAD51-GFP. Therefore, GFP fused to the C terminus of RAD51 abolishes the homologous pairing activity of RAD51 by disrupting secondary DNA binding, but does not affect its DMC1-stimulating activity.

    Original languageEnglish
    Pages (from-to)169-179
    Number of pages11
    JournalGenes and Genetic Systems
    Volume89
    Issue number4
    Publication statusPublished - 2014

    Fingerprint

    Green Fluorescent Proteins
    DNA
    DNA Repair
    Recombinational DNA Repair
    Chromosome Segregation
    Proteins
    Nucleoproteins
    Single-Stranded DNA
    Homologous Recombination
    Meiosis
    Arabidopsis
    Protein Isoforms
    Hydrolysis
    Adenosine Triphosphate

    Keywords

    • DMC1
    • GFP
    • Homologous pairing
    • Homologous recombination
    • RAD51

    ASJC Scopus subject areas

    • Genetics
    • Molecular Biology

    Cite this

    Green fluorescent protein fused to the C terminus ofRAD51 specifically interferes with secondary DNAbinding by the RAD51-ssDNA complex. / Kobayashi, Wataru; Sekine, Satoshi; Machida, Shinichi; Kurumizaka, Hitoshi.

    In: Genes and Genetic Systems, Vol. 89, No. 4, 2014, p. 169-179.

    Research output: Contribution to journalArticle

    Kobayashi, Wataru ; Sekine, Satoshi ; Machida, Shinichi ; Kurumizaka, Hitoshi. / Green fluorescent protein fused to the C terminus ofRAD51 specifically interferes with secondary DNAbinding by the RAD51-ssDNA complex. In: Genes and Genetic Systems. 2014 ; Vol. 89, No. 4. pp. 169-179.
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