Generation of versatile ss-dsDNA hybrid substrates for single-molecule analysis

Beláň, Ondrej; Moore, G. G. I.; Kaczmarczyk, Artur; Newton, Matthew D.; Anand, Roopesh; Boulton, Simon J.; Rueda, David

doi:10.1016/j.xpro.2021.100588

Cited by 11 publications

(10 citation statements)

References 16 publications

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“…To characterise the mechanical stability of the junction-containing DNA, we assembled a dumbbell by immobilising the DNA between two optically trapped beads using a microfluidics cell (Fig. 1B, C ) 20 , 30 , 31 . This dumbbell enables us to apply mechanical stretching force along the length of the DNA.…”

Section: Resultsmentioning

confidence: 99%

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

et al. 2022

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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.

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Section: Resultsmentioning

confidence: 99%

“…Single-molecule experiments were performed using a C-Trap (Lumicks) with integrated confocal microscopy and microfluidics, as described 20 , 30 , 31 . Prior to the experiment, all buffers were filtered with 0.2 μm syringe filters and microfluidic channels in the flow chamber (Fig.…”

Section: Methodsmentioning

confidence: 99%

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

et al. 2022

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“…To characterise the mechanical stability of the junctioncontaining DNA, we assembled a dumbbell by immobilising the DNA between two optically trapped beads using a microfluidics cell (Fig. 1B, C) 20,30,31 . This dumbbell enables us to apply mechanical stretching force along the length of the DNA.…”

Section: Resultsmentioning

confidence: 99%

Search and Processing of Holliday Junctions within Long DNA by Junction-Resolving Enzymes

Kaczmarczyk

Déclais

Newton

et al. 2022

Preprint

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Resolution of four-way Holliday junctions is a critical intermediate step of homologous recombination. DNA junctions must be processed by abundant junction-resolving endonucleases to cleave the covalent link between two chromosomes. Although the catalytic activity and interaction mode of endonucleases with Holliday junction have been characterized in great detail by biochemical and structural studies, it remains unclear how the enzymes find their substrate located within kilobase pairs of duplex DNA. Here, we present a complete single-molecule reaction trajectory for a junction-resolving enzyme finding and cleaving a Holliday junction. We employed correlative optical tweezers with confocal fluorescence microscopy to track a single dimer of endonuclease I on the DNA template in real-time. We observed that the enzyme binds remotely to the dsDNA and then undergoes 1D diffusion. Upon encountering the four-way junction, a catalytically impaired endonuclease I mutant remains bound at that point for long periods. An active enzyme, however, cleaves the junction after a few seconds. When an N-terminal truncated endonuclease mutant is used, the enzyme fails to diffuse along dsDNA and dissociates after short periods of time, indicating that DNA encirclement by the disordered N-terminus is a key requirement for 1D diffusion and efficient target localisation. Quantitative analysis of each of these stages revealed 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 likely to be applicable to most junction resolving enzymes.

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“…The workflow for preparing substrates with modified DNA inserts (Fig. 8E) is presented in Liu et al, 2015. Additional strategies for preparing custom DNA substrates can be found in Belan et al, 2021;Kim et al, 2017;Mueller et al, 2020.…”

Section: Preparing Biotinylated Dna Substratesmentioning

confidence: 99%

Visualizing the Dynamics of DNA Replication and Repair at the Single-Molecule Molecule Level

Chistol

Berger

2022

Preprint

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During cell division, the genome of each eukaryotic cell is copied by thousands of replisomes - large protein complexes consisting of several dozen proteins. Recent studies suggest that the eukaryotic replisome is much more dynamic than previously thought. To directly visualize replisome dynamics in a physiological context, we recently developed a single-molecule approach for imaging replication proteins in Xenopus egg extracts. These extracts contain all the soluble nuclear proteins and faithfully recapitulate DNA replication and repair in vitro, serving as a powerful platform for studying the mechanisms of genome maintenance. Here we present detailed protocols for conducting single-molecule experiments in nuclear egg extracts and preparing key reagents. This workflow can be easily adapted to visualize the dynamics and function of other proteins implicated in DNA replication and repair.

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Generation of versatile ss-dsDNA hybrid substrates for single-molecule analysis

Cited by 11 publications

References 16 publications

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

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

Search and Processing of Holliday Junctions within Long DNA by Junction-Resolving Enzymes

Visualizing the Dynamics of DNA Replication and Repair at the Single-Molecule Molecule Level

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