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

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posted on 2022-10-10, 10:32 authored by Artur P Kaczmarczyk, Anne-Cécile Déclais, Matthew D Newton, Simon J Boulton, David MJ Lilley, David S Rueda
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.

Funding

Crick (Grant ID: 10048, Grant title: Boulton FC001048) Wellcome Trust (Grant ID: 206292/Z/17/Z, Grant title: WT 206292/Z/17/Z) European Research Council (Grant ID: 742437 - TelMetab, Grant title: ERC 742437 - TelMetab)

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