Live cell imaging reveals that RecA finds homologous DNA by reduced dimensionality search

Abstract

The search for a homologous template is a central and critical step in the repair of DNA breaks by homologous recombination. However, it is still unclear how the search process is carried out within a cell. Here, we image double-stranded break (DSB) repair in living E. coli growing in a microfluidic device and show that two segregated homologous sequences find each other less than 9 min after an induced DSB. To characterize the mechanisms of the search process, we use a new RecA fluorescent fusion that rapidly forms structures after DSBs. Initially, RecA forms a bright cluster at the site of the DNA damage and then extends to form a thin, flexible, and dynamic filament. Based on our observations, we propose a model where the 1D ssDNA-RecA filament stretches along the cell, and the homology search is mediated by diffusion of the repair template that can interact with any segment of the filament. This model reduces the complexity of the search from 3D to 2D and quantitatively predicts that genome-wide search for homology can be finished in minutes, in agreement with our measurements.

SMC protein RecN drives RecA filament translocation and remodelling for in vivo homology search

“…We now characterize the dynamics of RecA during the homology search phase. In contrast to the static structures previously reported in E. coli (5,6), we find that once RecA nucleates at the DSB site near the pole, it rapidly grows into an elongated filament, following which it dynamically moves across the length of the cell (Fig. 1B,C, S2A, S2D; Supplementary Video 1).…”

“…It is possible that there is some diversity in RecA-dependent long-range homology search dynamics across bacterial systems, to contend with the varying cell shapes, chromosome replication dynamics (multi-fork vs replication only once per cell cycle), and chromosome organization (3)(4)(5)(6). However, given that RecA and RecN are among the most conserved proteins of the recombination pathway in bacterial genomes and its counterparts are present across domains of life, we believe that the dynamics we describe here is active in other systems as well.…”

“…Homologous recombination, a process that usually ensures error-free repair of DSBs has emerged as an evolutionarily conserved pathway from bacteria to eukaryotic cells (1,2). Recent work has shown that recombination-based repair is not only local, such as during post-replicative cohesion, but that even distant homologous regions can participate in such repair (2)(3)(4)(5)(6). This is particularly relevant for bacteria, where an extensive post-cohesion period is absent.…”

“…In vitro studies have shown that RecA molecules in the nucleoprotein filament possess ATPase activity that can influence (a) formation of a continuous filament during RecA loading on ssDNA (13), (b) growth and shrinkage of the filament (14-16), (c) release of the filament from heterologous pairing (9,17), and (d) RecA turnover during strand invasion (16)(17)(18). In vivo imaging of RecA in E. coli has revealed the presence of large assemblies of RecA, described as RecA filaments (or 'bundles'), that can even span the length of the cell (5,6).…”

Distribution of Holliday junctions and repair forks during Escherichia coli DNA double-strand break repair

“…A potential reason for initiation of recombination via paranemic invasion by a region on the single-strand coated with RecA protein is provided in a recent model of homology searching [ 43 ]. This model proposes that a single-strand of a broken chromosome, coated with RecA protein is extended across the E .…”