Search and processing of Holliday junctions within long DNA by junction-resolving enzymes

Artur P. Kaczmarczyk, Anne-Cécile Déclais, Matthew D. Newton, Simon J. Boulton, David M. J. Lilley (Lead / Corresponding author), David S. Rueda (Lead / Corresponding author)

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Resolution of Holliday junctions is a critical intermediate step of homologous recombination in which junctions are processed by junction-resolving endonucleases. Although binding and cleavage are well understood, the question remains how the enzymes locate their substrate within long duplex DNA. Here we track fluorescent dimers of endonuclease I on DNA, presenting the complete single-molecule reaction trajectory for a junction-resolving enzyme finding and cleaving a Holliday junction. We show that the enzyme binds remotely to dsDNA and then undergoes 1D diffusion. Upon encountering a four-way junction, a catalytically-impaired mutant remains bound at that point. An active enzyme, however, cleaves the junction after a few seconds. Quantitative analysis provides a comprehensive description of the facilitated diffusion mechanism. We show that the eukaryotic junction-resolving enzyme GEN1 also undergoes facilitated diffusion on dsDNA until it becomes located at a junction, so that the general resolution trajectory is probably applicable to many junction resolving enzymes.

Original languageEnglish
Article number5921
Number of pages13
JournalNature Communications
Publication statusPublished - 7 Oct 2022


  • DNA/metabolism
  • DNA, Cruciform
  • Deoxyribonuclease I/metabolism
  • Endodeoxyribonucleases/metabolism
  • Endonucleases/metabolism
  • Holliday Junction Resolvases/metabolism
  • Nucleic Acid Conformation

ASJC Scopus subject areas

  • General
  • Physics and Astronomy(all)
  • Chemistry(all)
  • Biochemistry, Genetics and Molecular Biology(all)


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