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

Abstract

While the molecular repertoire of the homologous recombination pathways is well studied, the search mechanism that enables recombination between distant homologous regions is poorly understood. Earlier work suggests that the recombinase RecA, an essential component for homology search, forms an elongated filament, nucleating at the break site. How this RecA structure carries out long-distance search remains unclear. Here, we follow the dynamics of RecA after induction of a single double-strand break on the Caulobacter chromosome. We find that the RecA-nucleoprotein filament, once formed, rapidly translocates in a directional manner in the cell, undergoing several pole-to-pole traversals, until homology search is complete. Concomitant with translocation, we observe dynamic variation in the length of the filament. Importantly in vivo, the RecA filament alone is incapable of such long-distance movement; both translocation and associated length variations are contingent on action of structural maintenance of chromosome (SMC)-like protein RecN, via its ATPase cycle. In summary, we have uncovered the three key elements of homology search driven by RecN: mobility of a finite segment of RecA, changes in filament length, and ability to conduct multiple pole-to-pole traversals, which together point to an optimal search strategy.

Mechanism of homology search expansion during recombinational DNA break repair

“…Distant ssDNA sites are thus spatially associated and at a null distance from the resection front in the absence of Rad51. These observations show that Rad51 extends the ssDNA molecule in WT cells, which otherwise collapses upon RPA binding in rad51Δ cells ( Figure 3E ), an interpretation consistent with globular RPA-ssDNA structures and elongated Rad51/RecA-ssDNA structures observed in vitro and in cells 14,16,18–21,48,50–54 . Consequently, stiffening of ssDNA by Rad51 projects DSB-proximal sites away from the DSB-borne chromatin environment, which promotes genome-wide homology search ( Figure 3E ).…”

“…A conserved property of RecA-family proteins is to polymerize on DNA into elongated structures exhibiting persistence length orders of magnitude greater than protein-free ssDNA in vitro 2 . Such structures were recently observed in bacterial and S. cerevisiae cells using fluorescently-tagged RecA/Rad51 proteins 18,20,21 . Our ssHi-C data and modeling support the proposal from these cytologic studies that the possibility of genome-wide homology search lies in NPF’s atypical, rigid structure.…”

“…A conserved property of RecA/Rad51-ssDNA NPFs is to adopt an elongated structure in vitro, with persistent length exceeding that of ssDNA by two to three orders of magnitude [14][15][16][17] . Recent cytological observations with functional or recessive fluorescently-labeled RecA/Rad51 proteins supported the existence of similar, micrometer-long, stiff fiber-like structures in cells, which led to diverse models of homology search promotion in bacteria and eukaryotes [18][19][20][21] . E.coli RecA-ssDNA NPFs stretching across the tubular nucleoid were proposed to reduce search complexity from 3D to 2D 18 , while the search of comparatively shorter C. crescentus NPFs was promoted by cohesin-like RecN-mediated movements, presumably anchored within the RecA-ssDNA NPF 21 .…”

“…Recent cytological observations with functional or recessive fluorescently-labeled RecA/Rad51 proteins supported the existence of similar, micrometer-long, stiff fiber-like structures in cells, which led to diverse models of homology search promotion in bacteria and eukaryotes [18][19][20][21] . E.coli RecA-ssDNA NPFs stretching across the tubular nucleoid were proposed to reduce search complexity from 3D to 2D 18 , while the search of comparatively shorter C. crescentus NPFs was promoted by cohesin-like RecN-mediated movements, presumably anchored within the RecA-ssDNA NPF 21 . In S. cerevisiae, dynamic elongation and collapse of Rad51 structures were proposed to optimize exploration of a spherical nucleus 20 .…”

Features of the DNAEscherichia coliRecN interaction revealed by fluorescence microscopy and single-molecule methods

“…The key function of the SMC-like protein RecN is the displacement of the RecA inside the cell through its ATPase activity. It has been shown that in the absence of the RecN, the RecA filament is not able to significantly move within the cell, and also the removal of the ATPase activity of RecN leads to blocking the movement of RecA into bacteria [6]. It is also known that RecN stimulates the ligation of linear duplex DNA molecules [7] and stimulates RecA-mediated DNA strand exchange [8].…”