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Force and ATP hydrolysis dependent regulation of RecA nucleoprotein filament by single-stranded DNA binding protein.

ABSTRACT: In Escherichia coli, the filament of RecA formed on single-stranded DNA (ssDNA) is essential for recombinational DNA repair. Although ssDNA-binding protein (SSB) plays a complicated role in RecA reactions in vivo, much of our understanding of the mechanism is based on RecA binding directly to ssDNA. Here we investigate the role of SSB in the regulation of RecA polymerization on ssDNA, based on the differential force responses of a single 576-nucleotide-long ssDNA associated with RecA and SSB. We find that SSB outcompetes higher concentrations of RecA, resulting in inhibition of RecA nucleation. In addition, we find that pre-formed RecA filaments de-polymerize at low force in an ATP hydrolysis- and SSB-dependent manner. At higher forces, re-polymerization takes place, which displaces SSB from ssDNA. These findings provide a physical picture of the competition between RecA and SSB under tension on the scale of the entire nucleoprotein SSB array, which have broad biological implications particularly with regard to competitive molecular binding.

SUBMITTER: Fu H

PROVIDER: S-EPMC3553936 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Organized unidirectional waves of ATP hydrolysis within a RecA filament.

Project description:The RecA protein forms nucleoprotein filaments on DNA, and individual monomers within the filaments hydrolyze ATP. Assembly and disassembly of filaments are both unidirectional, occurring on opposite filament ends, with disassembly requiring ATP hydrolysis. When filaments form on duplex DNA, RecA protein exhibits a functional state comparable to the state observed during active DNA strand exchange. RecA filament state was monitored with a coupled spectrophotometric assay for ATP hydrolysis, with changes fit to a mathematical model for filament disassembly. At 37 degrees C, monomers within the RecA-double-stranded DNA (dsDNA) filaments hydrolyze ATP with an observed k(cat) of 20.8 +/- 1.5 min(-1). Under the same conditions, the rate of end-dependent filament disassembly (k(off)) is 123 +/- 16 monomers per minute per filament end. This rate of disassembly requires a tight coupling of the ATP hydrolytic cycles of adjacent RecA monomers. The relationship of k(cat) to k(off) infers a filament state in which waves of ATP hydrolysis move unidirectionally through RecA filaments on dsDNA, with successive waves occurring at intervals of approximately six monomers. The waves move nearly synchronously, each one transiting from one monomer to the next every 0.5 s. The results reflect an organization of the ATPase activity that is unique in filamentous systems, and could be linked to a RecA motor function.

| S-EPMC546331 | biostudies-literature

RecA-SSB Interaction Modulates RecA Nucleoprotein Filament Formation on SSB-Wrapped DNA.

Project description:E. coli RecA recombinase catalyzes the homology pairing and strand exchange reactions in homologous recombinational repair. RecA must compete with single-stranded DNA binding proteins (SSB) for single-stranded DNA (ssDNA) substrates to form RecA nucleoprotein filaments, as the first step of this repair process. It has been suggested that RecA filaments assemble mainly by binding and extending onto the free ssDNA region not covered by SSB, or are assisted by mediators. Using the tethered particle motion (TPM) technique, we monitored individual RecA filament assembly on SSB-wrapped ssDNA in real-time. Nucleation times of the RecA E38K nucleoprotein filament assembly showed no apparent dependence among DNA substrates with various ssDNA gap lengths (from 60 to 100 nucleotides) wrapped by one SSB in the (SSB)65 binding mode. Our data have shown an unexpected RecA filament assembly mechanism in which a RecA-SSB-ssDNA interaction exists. Four additional pieces of evidence support our claim: the nucleation times of the RecA assembly varied (1) when DNA substrates contained different numbers of bound SSB tetramers; (2) when the SSB wrapping mode conversion is induced; (3) when SSB C-terminus truncation mutants are used; and (4) when an excess of C-terminal peptide of SSB is present. Thus, a RecA-SSB interaction should be included in discussing RecA regulatory mechanism.

| S-EPMC5605508 | biostudies-literature

A 5'-to-3' strand exchange polarity is intrinsic to RecA nucleoprotein filaments in the absence of ATP hydrolysis.

Project description:RecA is essential to recombinational DNA repair in which RecA filaments mediate the homologous DNA pairing and strand exchange. Both RecA filament assembly and the subsequent DNA strand exchange are directional. Here, we demonstrate that the polarity of DNA strand exchange is embedded within RecA filaments even in the absence of ATP hydrolysis, at least over short DNA segments. Using single-molecule tethered particle motion, we show that successful strand exchange in the presence of ATP proceeds with a 5'-to-3' polarity, as demonstrated previously. RecA filaments prepared with ATPγS also exhibit a 5'-to-3' progress of strand exchange, suggesting that the polarity is not determined by RecA disassembly and/or ATP hydrolysis. RecAΔC17 mutants, lacking a C-terminal autoregulatory flap, also promote strand exchange in a 5'-to-3' polarity in ATPγS, a polarity that is largely lost with this RecA variant when ATP is hydrolyzed. We propose that there is an inherent strand exchange polarity mediated by the structure of the RecA filament groove, associated by conformation changes propagated in a polar manner as DNA is progressively exchanged. ATP hydrolysis is coupled to polar strand exchange over longer distances, and its contribution to the polarity requires an intact RecA C-terminus.

| S-EPMC6547424 | biostudies-literature

Single-Molecule Insights into ATP-Dependent Conformational Dynamics of Nucleoprotein Filaments of Deinococcus radiodurans RecA.

Project description:Deinococcus radiodurans (Dr) has one of the most robust DNA repair systems, which is capable of withstanding extreme doses of ionizing radiation and other sources of DNA damage. DrRecA, a central enzyme of recombinational DNA repair, is essential for extreme radioresistance. In the presence of ATP, DrRecA forms nucleoprotein filaments on DNA, similar to other bacterial RecA and eukaryotic DNA strand exchange proteins. However, DrRecA catalyzes DNA strand exchange in a unique reverse pathway. Here, we study the dynamics of DrRecA filaments formed on individual molecules of duplex and single-stranded DNA, and we follow conformational transitions triggered by ATP hydrolysis. Our results reveal that ATP hydrolysis promotes rapid DrRecA dissociation from duplex DNA, whereas on single-stranded DNA, DrRecA filaments interconvert between stretched and compressed conformations, which is a behavior shared by E. coli RecA and human Rad51. This indicates a high conservation of conformational switching in nucleoprotein filaments and suggests that additional factors might contribute to an inverse pathway of DrRecA strand exchange.

| S-EPMC7583915 | biostudies-literature

Stable Nuclei of Nucleoprotein Filament and High ssDNA Binding Affinity Contribute to Enhanced RecA E38K Recombinase Activity.

Project description:RecA plays central roles in the homologous recombination to repair double-stranded DNA break damage in E. coli. A previously identified recA strain surviving high doses of UV radiation includes a dominant RecA E38K mutation. Using single-molecule experiments, we showed that the RecA E38K variant protein assembles nucleoprotein filaments more rapidly than the wild-type RecA. We also used a single-molecule fluorescence resonance energy transfer (smFRET) experiment to compare the nucleation cluster dynamics of wild-type RecA and RecA E38K mutants on various short ssDNA substrates. At shorter ssDNA, nucleation clusters of RecA E38K form dynamically, while only few were seen in wild-type RecA. RecA E38K also forms stable nuclei by specifically lowering the dissociation rate constant, k d . These observations provide evidence that greater nuclei stability and higher ssDNA binding affinity contribute to the observed enhanced recombination activity of the RecA E38K mutant. Given that assembly of RecA nucleoprotein filaments is the first committed step in recombinational repair processes, enhancement at this step gives rise to a more efficient recombinase.

| S-EPMC5668366 | biostudies-literature

RAD51 protein ATP cap regulates nucleoprotein filament stability.

Project description:RAD51 mediates homologous recombination by forming an active DNA nucleoprotein filament (NPF). A conserved aspartate that forms a salt bridge with the ATP ?-phosphate is found at the nucleotide-binding interface between RAD51 subunits of the NPF known as the ATP cap. The salt bridge accounts for the nonphysiological cation(s) required to fully activate the RAD51 NPF. In contrast, RecA homologs and most RAD51 paralogs contain a conserved lysine at the analogous structural position. We demonstrate that substitution of human RAD51(Asp-316) with lysine (HsRAD51(D316K)) decreases NPF turnover and facilitates considerably improved recombinase functions. Structural analysis shows that archaebacterial Methanococcus voltae RadA(D302K) (MvRAD51(D302K)) and HsRAD51(D316K) form extended active NPFs without salt. These studies suggest that the HsRAD51(Asp-316) salt bridge may function as a conformational sensor that enhances turnover at the expense of recombinase activity.

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RecA filament maintains structural integrity using ATP-driven internal dynamics.

Project description:At the core of homologous DNA repair, RecA catalyzes the strand exchange reaction. This process is initiated by a RecA loading protein, which nucleates clusters of RecA proteins on single-stranded DNA. Each cluster grows to cover the single-stranded DNA but may leave 1- to 2-nucleotide (nt) gaps between the clusters due to three different structural phases of the nucleoprotein filaments. It remains to be revealed how RecA proteins eliminate the gaps to make a seamless kilobase-long filament. We develop a single-molecule fluorescence assay to observe the novel internal dynamics of the RecA filament. We directly observe the structural phases of individual RecA filaments and find that RecA proteins move their positions along the substrate DNA to change the phase of the filament. This reorganization process, which is a prerequisite step for interjoining of two adjacent clusters, requires adenosine triphosphate hydrolysis and is tightly regulated by the recombination hotspot, Chi. Furthermore, RecA proteins recognize and self-align to a 3-nt-period sequence pattern of TGG. This sequence-dependent phase bias may help the RecA filament to maintain structural integrity within the kilobase-long filament for accurate homology search and strand exchange reaction.

| S-EPMC5587095 | biostudies-literature

SSB protein diffusion on single-stranded DNA stimulates RecA filament formation.

Project description:Single-stranded DNA generated in the cell during DNA metabolism is stabilized and protected by binding of ssDNA-binding (SSB) proteins. Escherichia coli SSB, a representative homotetrameric SSB, binds to ssDNA by wrapping the DNA using its four subunits. However, such a tightly wrapped, high-affinity protein-DNA complex still needs to be removed or repositioned quickly for unhindered action of other proteins. Here we show, using single-molecule two- and three-colour fluorescence resonance energy transfer, that tetrameric SSB can spontaneously migrate along ssDNA. Diffusional migration of SSB helps in the local displacement of SSB by an elongating RecA filament. SSB diffusion also melts short DNA hairpins transiently and stimulates RecA filament elongation on DNA with secondary structure. This observation of diffusional movement of a protein on ssDNA introduces a new model for how an SSB protein can be redistributed, while remaining tightly bound to ssDNA during recombination and repair processes.

| S-EPMC2782680 | biostudies-literature

Structural and Functional Studies of H. seropedicae RecA Protein - Insights into the Polymerization of RecA Protein as Nucleoprotein Filament.

Project description:The bacterial RecA protein plays a role in the complex system of DNA damage repair. Here, we report the functional and structural characterization of the Herbaspirillum seropedicae RecA protein (HsRecA). HsRecA protein is more efficient at displacing SSB protein from ssDNA than Escherichia coli RecA protein. HsRecA also promotes DNA strand exchange more efficiently. The three dimensional structure of HsRecA-ADP/ATP complex has been solved to 1.7 Å resolution. HsRecA protein contains a small N-terminal domain, a central core ATPase domain and a large C-terminal domain, that are similar to homologous bacterial RecA proteins. Comparative structural analysis showed that the N-terminal polymerization motif of archaeal and eukaryotic RecA family proteins are also present in bacterial RecAs. Reconstruction of electrostatic potential from the hexameric structure of HsRecA-ADP/ATP revealed a high positive charge along the inner side, where ssDNA is bound inside the filament. The properties of this surface may explain the greater capacity of HsRecA protein to bind ssDNA, forming a contiguous nucleoprotein filament, displace SSB and promote DNA exchange relative to EcRecA. Our functional and structural analyses provide insight into the molecular mechanisms of polymerization of bacterial RecA as a helical nucleoprotein filament.

| S-EPMC4957752 | biostudies-literature

RecA polymerization on double-stranded DNA by using single-molecule manipulation: the role of ATP hydrolysis.

Project description:The polymerization of RecA on individual double-stranded DNA molecules is studied. A linear DNA (lambda DNA, 48.5 Kb), anchored at one end to a cover glass and at the other end to an optically trapped 3-micrometers diameter polystyrene bead, serves as a template. The elongation caused by RecA assembly is measured in the presence of ATP and ATP[gammaS]. By using force extension and hydrodynamic recoil, a value of the persistence length of the RecA-DNA complex is obtained. In the presence of ATP, the polymer length is unstable, first growing to saturation and then decreasing. This suggests a transient dynamics of association and dissociation for RecA on a double-stranded DNA, the process being controlled by ATP hydrolysis. Part of this dynamics is suppressed in the presence of ATP[gammaS], leading to a stabilized RecA-DNA complex. A one-dimensional nucleation and growth model is presented that may account for the protein assembly.

| S-EPMC22162 | biostudies-other

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