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

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.

Visualization of direct and diffusion-assisted RAD51 nucleation by full-length human BRCA2 protein

“…Recently, other proteins, such as endonuclease I, were observed to bind and diffuse on dsDNA despite apparent low dsDNA affinity. 35 BRCA2-RAD51 complexes move mostly on dsDNA, whereas they display static behavior on ssDNA ( Figure 4B ). In the presence of RPA, slightly less than half of RAD51 nucleation events at the ssDNA gaps result from RAD51-BRCA2 molecules that reach the ssDNA gap by moving along the dsDNA arms ( Figure 4C ).…”

“…To understand the movement of BRCA2 on dsDNA in more detail, we extracted the trajectory of fluorescent molecules using a custom-written single-particle tracking algorithm 35 and then calculated the mean square displacement (MSD) of moving BRCA2-RAD51 complexes. Proteins can move on DNA via several mechanisms, including free 1D diffusion (random walk), constrained diffusion (where diffusion barriers are present), and directed motion (e.g., ATPase-driven translocation).…”

“…Owing to DNA shielding by cations, hopping is expected to increase the diffusion coefficient as a function of salt concentration, whereas sliding does not. 35 , 42 , 43 We probed the diffusion of the BRCA2-RAD51 complex in the presence of 25–150 mM NaCl ( Figure 5B ). Increasing salt concentrations decreased the overall DNA-binding frequency of BRCA2 ( Figure 5C ) but did not increase the diffusion coefficient.…”

“…For the mean square displacement (MSD) analysis, we used a custom-made single-particle tracking algorithm in Python, 35 which was incorporated within each Jupiter Notebook workspace. The kymograph imported from an.h5 file was cropped to eliminate the oversaturated parts of the image (i.e., the bead contours).…”

A Biophysics Toolbox for Reliable Data Acquisition and Processing in Integrated Force–Confocal Fluorescence Microscopy

“…An additional advantage of dual-beam OT over MT and single-beam OT is that instead of being tethered on the surface at one end, the molecules of interest are separated from the surface by several micrometers, effectively inhibiting undesirable surface adhesion. Taken together, dual-beam OT force-fluorescence spectroscopy has become a powerful single-molecule biophysics technique, broadly used to investigate a number of biological processes including, but not limited to, DNA replication, DNA repair, and protein folding dynamics. ,− The focus of this method article is the application of dual-beam OT force–fluorescence spectroscopy in studying protein:DNA interactions, one of the fields that have benefited most from the development of this approach.…”