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

Abstract

RecA protein mediates homologous recombination repair in bacteria through assembly of long helical filaments on single-stranded DNA (ssDNA) in an ATP dependent manner. RecX, an important negative regulator of RecA, is known to inhibit RecA activity by stimulating the disassembly of RecA nucleoprotein filaments. Here we use a single-molecule approach to address the regulation of (E. coli) RecA-ssDNA filaments by RecX (E. coli) within the framework of distinct conformational states of RecA-ssDNA filament. Our findings revealed that RecX effectively binds the inactive conformation of RecA-ssDNA filaments and slows down the transition to the active state. Results of this work provide new mechanistic insights into the RecX-RecA interactions and highlight the importance of conformational transitions of RecA filaments as an additional level of regulation of its biological activity.

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

“…Optical trapping experiments were performed to visualize RecN::mCherry protein binding to individual double-stranded and single-stranded DNA molecules. DNA manipulation was carried out inside a five-channel microfluidic system (uFlux, Lumicks), using two polystyrene microspheres coated with streptavidin (Spherotech, 2.1 μm diameter) and held by two optical traps according to the method presented in [15,16]. Double-stranded linear DNA molecules ∼23 kb length with biotinylated ends and obtained by modifying the DNA of bacteriophage lambda were initially used as a DNA substrate.…”

Properties of theUreaplasma parvumSMC protein related to its interaction with DNA

“…Optical trapping experiments were performed to analyze SMC protein binding to individual double-stranded and single-stranded DNA molecules. DNA manipulation was carried out inside a five-channel microfluidic system (uFlux, Lumicks), using two polystyrene microspheres coated with streptavidin (Spherotech, 2.1 μm diameter) and held by two optical traps according to the method presented in [20, 21]. A 11070 bp long linear DNA molecule (prlC) was used as substrate.…”

Modeling the Homologous Recombination Process: Methods, Successes and Challenges

“…The consequences of the switch in binding geometries are significant in terms of accessibility of the amino acids, electrostatic properties or topology of the filament deep groove [20]. The topology of the groove, including its width, the presence of longitudinal tracks and more generally the partitioning of the 3D space, rules the relative positions of the DNA strands [50] but also the access of partner proteins to the filament interior; for example, the LexA repressor protein only binds the stretched form of RecA filament which catalyzes its self-cleavage [113], while RecX regulates the HR process by specifically binding to the compressed form [13,114].…”

“…Most of these proteins bind in the filament deep groove, hence the importance of the filament topology and plasticity. For example, RecX specifically binds filaments in the compressed form while LexA or DinI bind filaments in the stretched form [13,114]. Proteins that participate to the control of the HR reaction often bind filament via unstructured regions, making the construction of models a challenging task.…”

“…HR filaments catalyze ATP hydrolysis and ATP is continuously consumed in great quantities during the whole HR process [11]. ATP hydrolysis has been associated with polymorphism of the HR filament [12]; the filament plasticity is proposed to play a role in the regulation of the HR process notably by favoring dsDNA unbinding [10,13,14], but this role still needs to be fully characterized. Finally, while in vivo the HR process is tuned or controlled by several factors, such as the salt concentration (magnesium in bacteria [15,16], calcium in higher organisms [17]) or the interaction with auxiliary proteins [3]; in vitro studies showed that most HR steps can happen in the absence of auxiliary proteins, highlighting the specific role of the nucleoprotein filaments in the HR process; all HR steps take place within these evolving complexes and therefore benefit from their structural, mechanical and dynamical properties [4,18].…”