Single‐molecule analysis reveals two distinct states of the compressed RecA filament on single‐stranded DNA

Abstract

The RecA protein plays a key role in bacterial homologous recombination (HR) and acts through assembly of long helical filaments around single‐stranded DNA in the presence of ATP. Large‐scale conformational changes induced by ATP hydrolysis result in transitions between stretched and compressed forms of the filament. Here, using a single‐molecule approach, we show that compressed RecA nucleoprotein filaments can exist in two distinct interconvertible states depending on the presence of ADP in the monomer–monomer interface. Binding of ADP promotes cooperative conformational transitions and directly affects mechanical properties of the filament. Our findings reveal that RecA nucleoprotein filaments are able to continuously cycle between three mechanically distinct states that might have important implications for RecA‐mediated processes of HR.

A new insight into RecA filament regulation by RecX from the analysis of conformation-specific interactions

“…At a stretching force of 3 pN the length of the RecA-ssDNA filament is significantly greater than the length of bare ssDNA [6,18] (Figure S1). Therefore, shortening of the RecA-ssDNA tether can be caused by RecX actively displacing RecA from ssDNA.…”

“…Corresponding dependence is presented in figure 1E and is well fitted by a Hill equation with a Hill coefficient of 2.0 ± 0. At a stretching force of 3 pN the length of the RecA-ssDNA filament is significantly greater than the length of bare ssDNA [6,18] (Figure S1). 2A).…”

“…In this work we addressed the dynamics of interaction between E. coli RecX and RecA nucleoprotein filaments (Figure 1A) utilizing a single-molecule approach which combines dual-trap optical tweezers and a microfluidic laminar flow cell for manipulation of individual DNA molecules (Figure 1B) as reported elsewhere [18,[42][43][44][45][46][47][48]. The microfluidic flow cell (Lumicks B.V.) consisted of 5 laminar flow channels which facilitated optical trapping of the beads, DNA tethering and RecA nucleoprotein filaments assembly (see Methods).…”

“…Importantly, RecA-dependent SOS-response activation is one of pathways for development of antibiotic resistance by bacteria through enhanced mutagenesis in the presence of continuous DNA damage. RecA nucleoprotein filaments are very dynamic, and may experience large-scale conformational changes induced by ATP binding and hydrolysis [6,[15][16][17][18]. Besides intrinsic ATPase activity of RecA, assembly and stability of RecA-ssDNA filaments are also dynamically regulated by a network of various protein mediators [19][20][21][22][23].…”

“…Recently, we characterized three mechanically distinct conformational states that occur within the E. coli RecA-ssDNA filaments in the course of ATP hydrolysis [18]. In the present work, building on the developed single-molecule assay we aimed to study interactions of E. coli RecX with different forms of RecA nucleoprotein filaments to further deepen understanding of RecX inhibitory mechanism.…”