Biochemical and Structural Properties of Fungal Holliday Junction-Resolving Enzymes

    Research output: Chapter in Book/Report/Conference proceedingChapter (peer-reviewed)peer-review

    1 Citation (Scopus)


    Four-way Holliday junctions in DNA are the central intermediates of genetic recombination and must be processed into regular duplex species. One mechanism for achieving this is called resolution, brought about by structure-selective nucleases. GEN1 is an important junction-resolving enzyme in eukaryotic cells, a member of the FEN1/EXO1 superfamily of nucleases. While human GEN1 is difficult to work with because of aggregation, orthologs from thermophilic fungi have been identified using bioinformatics and have proved to have excellent properties. Here, the expression and purification of this enzyme from Chaetomium thermophilum is described, together with the means of investigating its biochemical properties. The enzyme is quite similar to junction-resolving enzymes from lower organisms, binding to junctions in dimeric form, introducing symmetrical bilateral cleavages, the second of which is accelerated to promote productive resolution. Crystallization of C. thermophilum GEN1 is described, and the structure of a DNA-product complex. Juxtaposition of complexes in the crystal lattice suggests how the structure of a dimeric enzyme with an intact junction is organized.

    Original languageEnglish
    Title of host publicationMechanisms of DNA Recombination and Genome Rearrangements
    Subtitle of host publicationMethods to Study Homologous Recombination
    EditorsMaria Spies, Anna Malkova
    PublisherAcademic Press
    Number of pages26
    ISBN (Print)9780128144299
    Publication statusPublished - 2018

    Publication series

    NameMethods in Enzymology
    PublisherAcademic Press
    ISSN (Print)0076-6879


    • Bioinformatics
    • DNA repair
    • Enzyme kinetics
    • Four-way DNA junction
    • GEN1
    • Genetic recombination
    • Nucleases
    • X-ray crystallography

    ASJC Scopus subject areas

    • Biochemistry
    • Molecular Biology


    Dive into the research topics of 'Biochemical and Structural Properties of Fungal Holliday Junction-Resolving Enzymes'. Together they form a unique fingerprint.

    Cite this