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 () and David S. Rueda ()
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Artur P. Kaczmarczyk: Imperial College London
Anne-Cécile Déclais: University of Dundee
Matthew D. Newton: Imperial College London
Simon J. Boulton: The Francis Crick Institute
David M. J. Lilley: University of Dundee
David S. Rueda: Imperial College London

Nature Communications, 2022, vol. 13, issue 1, 1-13

Abstract: Abstract 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.

Date: 2022
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DOI: 10.1038/s41467-022-33503-6

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