Biochemical and Structural Properties of Fungal Holliday Junction-Resolving Enzymes

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Abstract

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
Pages543-568
Number of pages26
Volume600
ISBN (Print)9780128144299
DOIs
Publication statusPublished - 2018

Publication series

NameMethods in Enzymology
PublisherAcademic Press
Volume600
ISSN (Print)0076-6879

Keywords

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

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