Journal
NATURE COMMUNICATIONS
Volume 13, Issue 1, Pages -Publisher
NATURE PORTFOLIO
DOI: 10.1038/s41467-022-33503-6
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Funding
- MRC-London Institute of Medical Sciences [UKRI MC-A658-5TY10]
- Wellcome Trust [206292/Z/17/Z, FC0010048]
- BBSRC CASE-studentship
- Cancer Research UK [A18604]
- Francis Crick Institute - Cancer Research UK [FC0010048]
- UK Medical Research Council [FC0010048]
- European Research Council (ERC) Advanced Investigator Grant (TelMetab)
- Wellcome Trust
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This study captures the entire reaction trajectory of a nuclease targeting and resolving a DNA junction at single-molecule level. The results show that the enzyme first binds remotely to dsDNA and undergoes 1D diffusion before cleaving the junction upon encountering it.
Locating a four-way junction in a high background of genomic DNA is likely to be the rate-limiting step of the resolution process. This study captures the entire reaction trajectory of a nuclease targeting and resolving a DNA junction at single-molecule level. 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.
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