RecA Nucleoprotein Filament Research Articles

Studying RecA Homology Search Mechanism using Single-Molecule Methods

RecA recombinase plays an important role in repairing the double-strand DNA breaks in E. coli. To repair damaged DNA, RecA first forms a nucleoprotein filament on a single-stranded DNA molecule, this nucleoprotein filament then searches and pairs the homology duplex DNA, and finally carries out the strand exchange reaction to initiate the homologous recombinational repair. To ensure that the repair process is carried out within the limited timescale set by the cell cycle of E. coli, it is important that RecA nucleoprotein filament locates its homologous sequence efficiently. Several search mechanisms have been proposed: a one-dimensional sliding (1D), a three-dimensional hopping (3D), and a facilitated intersegment transfer model. We have used two single-molecule imaging techniques to investigate the homology search mechanism of RecA nucleoprotein filaments. By applying a lateral force of a few pN to a bead-tethered dsDNA, the dsDNA is extended horizontally, and the diffusion trajectories of the labeled RecA nucleoprotein filaments (labeled with a bead or quantum dot) can be visualized at the single-molecule level. From individual searching events, the diffusion coefficient of the RecA nucleoprotein filament is determined to be ∼ 106 nm2/s. The salt dependence of the diffusion coefficients from 0 to 200 mM NaCl allows us to distinguish its major search mechanism.

ATP Hydrolysis Dependent Dynamic Stability of RecA Nucleofilament on Double-Stranded DNA

The RecA nucleoprotein filament formed on double-stranded (ds) DNA exhibits a functional state comparable to the state observed during active DNA strand exchange. By stretching short DNA of only a few hundreds base pairs, we observed high-resolution saw-tooth dynamics of RecA polymerization and de-polymerization on a single dsDNA molecule that was unable to be seen in previous single-DNA stretching experiments when much longer DNA was used. Novel saw-tooth fluctuation of the nucleoprotein filament was observed, allowing us to monitor the real time competition between RecA on and off DNA strands driven by the ATP-hydrolysis at a nanometer resolution.

Characterizing an Intermediate State Between Inactive and Active States of RecA

RecA is an essential enzyme for the repair of DNA double-strand breaks by homologous recombination. The repair requires the formation of a RecA nucleoprotein filament, in which ATP and DNA are known to promote each other's binding to RecA. The RecA filament exists in two distinct conformations: a wide and compressed “inactive” conformation in the absence of DNA, and a narrow and extended “active” conformation in the presence of ATP and DNA. The inactive conformations share similar crystal structures, although biochemically only the ATP-bound state has a high DNA binding affinity. Our molecular dynamics (MD) simulations on inactive RecA filament reveal that upon ATP binding, only parts of the RecA filament adopt active-like conformations. During the simulations, the DNA binding loops assemble into an arrangement optimal structure for binding of extended ssDNA, i.e., similar to the active ssDNA-RecA state. However, the overall structure remains in its inactive form. This suggests that the ATP-bound RecA might be an intermediate state between the inactive apo-RecA and active ssDNA-RecA states. This intermediate shows overall compressed inactive conformation, with key loops in extended active-like conformation. In order to study the cooperativity between DNA and ATP, we have also performed MD simulations on nucleoprotein filament, showing that the ATP-bound state is able to maintain the organized DNA binding loops both with and without ssDNA. However, in the absence of ATP, the DNA binding loops tend to lose their organized arrangement rapidly once the DNA is removed, a process which is followed by the overall conformational changes of the protein.

Mycobacterium leprae RecA is structurally analogous but functionally distinct from Mycobacterium tuberculosis RecA protein

Mycobacterium leprae is closely related to Mycobacterium tuberculosis, yet causes a very different illness. Detailed genomic comparison between these two species of mycobacteria reveals that the decaying M. leprae genome contains less than half of the M. tuberculosis functional genes. The reduction of genome size and accumulation of pseudogenes in the M. leprae genome is thought to result from multiple recombination events between related repetitive sequences, which provided the impetus to investigate the recombination-like activities of RecA protein. In this study, we have cloned, over-expressed and purified M. leprae RecA and compared its activities with that of M. tuberculosis RecA. Both proteins, despite being 91% identical at the amino acid level, exhibit strikingly different binding profiles for single-stranded DNA with varying GC contents, in the ability to catalyze the formation of D-loops and to promote DNA strand exchange. The kinetics and the extent of single-stranded DNA-dependent ATPase and coprotease activities were nearly equivalent between these two recombinases. However, the degree of inhibition exerted by a range of ATP:ADP ratios was greater on strand exchange promoted by M. leprae RecA compared to its M. tuberculosis counterpart. Taken together, our results provide insights into the mechanistic aspects of homologous recombination and coprotease activity promoted by M. lepare RecA, and further suggests that it differs from the M. tuberculosis counterpart. These results are consistent with an emerging concept of DNA-sequence influenced structural differences in RecA nucleoprotein filaments and how these differences reflect on the multiple activities associated with RecA protein.

The Escherichia coli DinD Protein Modulates RecA Activity by Inhibiting Postsynaptic RecA Filaments

Escherichia coli dinD is an SOS gene up-regulated in response to DNA damage. We find that the purified DinD protein is a novel inhibitor of RecA-mediated DNA strand exchange activities. Most modulators of RecA protein activity act by controlling the amount of RecA protein bound to single-stranded DNA by affecting either the loading of RecA protein onto DNA or the disassembly of RecA nucleoprotein filaments bound to single-stranded DNA. The DinD protein, however, acts postsynaptically to inhibit RecA during an on-going DNA strand exchange, likely through the disassembly of RecA filaments. DinD protein does not affect RecA single-stranded DNA filaments but efficiently disassembles RecA when bound to two or more DNA strands, effectively halting RecA-mediated branch migration. By utilizing a nonspecific duplex DNA-binding protein, YebG, we show that the DinD effect is not simply due to duplex DNA sequestration. We present a model suggesting that the negative effects of DinD protein are targeted to a specific conformational state of the RecA protein and discuss the potential role of DinD protein in the regulation of recombinational DNA repair.

Developing Single-Molecule TPM Experiments for Direct Observation of Successful RecA-Mediated Strand Exchange Reaction

RecA recombinases play a central role in homologous recombination. Once assembled on single-stranded (ss) DNA, RecA nucleoprotein filaments mediate the pairing of homologous DNA sequences and strand exchange processes. We have designed two experiments based on tethered particle motion (TPM) to investigate the fates of the invading and the outgoing strands during E. coli RecA-mediated pairing and strand exchange at the single-molecule level in the absence of force. TPM experiments measure the tethered bead Brownian motion indicative of the DNA tether length change resulting from RecA binding and dissociation. Experiments with beads labeled on either the invading strand or the outgoing strand showed that DNA pairing and strand exchange occurs successfully in the presence of either ATP or its non-hydrolyzable analog, ATPγS. The strand exchange rates and efficiencies are similar under both ATP and ATPγS conditions. In addition, the Brownian motion time-courses suggest that the strand exchange process progresses uni-directionally in the 5′-to-3′ fashion, using a synapse segment with a wide and continuous size distribution.

Two Modes of Binding of DinI to RecA Filament Provide a New Insight into the Regulation of SOS Response by DinI Protein

RecA protein plays a principal role in bacterial SOS response to DNA damage. The induction of the SOS response is well understood and involves the cleavage of the LexA repressor catalyzed by the RecA nucleoprotein filament. In contrast, our understanding of the regulation and termination of the SOS response is much more limited. RecX and DinI are two major regulators of RecA's ability to promote LexA cleavage and strand exchange reaction, and are believed to modulate its activity in ongoing SOS events. DinI's function in the SOS response remains controversial, since its interaction with the RecA filament is concentration dependent and may result in either stabilization or depolymerization of the filament. The 17 C-terminal residues of RecA modulate the interaction between DinI and RecA. We demonstrate that DinI binds to the active RecA filament in two distinct structural modes. In the first mode, DinI binds to the C-terminus of a RecA protomer. In the second mode, DinI resides deeply in the groove of the RecA filament, with its negatively charged C-terminal helix proximal to the L2 loop of RecA. The deletion of the 17 C-terminal residues of RecA favors the second mode of binding. We suggest that the negatively charged C-terminus of RecA prevents DinI from entering the groove and protects the RecA filament from depolymerization. Polymorphic binding of DinI to RecA filaments implies an even more complex role of DinI in the bacterial SOS response.

RecA K72R Filament Formation Defects Reveal an Oligomeric RecA Species Involved in Filament Extension

Using an ensemble approach, we demonstrate that an oligomeric RecA species is required for the extension phase of RecA filament formation. The RecA K72R mutant protein can bind but not hydrolyze ATP or dATP. When mixed with other RecA variants, RecA K72R causes a drop in the rate of ATP hydrolysis and has been used to study disassembly of hydrolysis-proficient RecA protein filaments. RecA K72R filaments do not form in the presence of ATP but do so when dATP is provided. We demonstrate that in the presence of ATP, RecA K72R is defective for extension of RecA filaments on DNA. This defect is partially rescued when the mutant protein is mixed with sufficient levels of wild type RecA protein. Functional extension complexes form most readily when wild type RecA is in excess of RecA K72R. Thus, RecA K72R inhibits hydrolysis-proficient RecA proteins by interacting with them in solution and preventing the extension phase of filament assembly.

Creating Directed Double-strand Breaks with the Ref Protein: A NOVEL RecA-DEPENDENT NUCLEASE FROM BACTERIOPHAGE P1

The bacteriophage P1-encoded Ref protein enhances RecA-dependent recombination in vivo by an unknown mechanism. We demonstrate that Ref is a new type of enzyme; that is, a RecA-dependent nuclease. Ref binds to ss- and dsDNA but does not cleave any DNA substrate until RecA protein and ATP are added to form RecA nucleoprotein filaments. Ref cleaves only where RecA protein is bound. RecA functions as a co-nuclease in the Ref/RecA system. Ref nuclease activity can be limited to the targeted strands of short RecA-containing D-loops. The result is a uniquely programmable endonuclease activity, producing targeted double-strand breaks at any chosen DNA sequence in an oligonucleotide-directed fashion. We present evidence indicating that cleavage occurs in the RecA filament groove. The structure of the Ref protein has been determined to 1.4 Å resolution. The core structure, consisting of residues 77-186, consists of a central 2-stranded β-hairpin that is sandwiched between several α-helical and extended loop elements. The N-terminal 76 amino acid residues are disordered; this flexible region is required for optimal activity. The overall structure of Ref, including several putative active site histidine residues, defines a new subclass of HNH-family nucleases. We propose that enhancement of recombination by Ref reflects the introduction of directed, recombinogenic double-strand breaks.

Dual Molecule Assay to Investigate the Mechanics of Homology Search and Recognition in Recombination

Central to homologous recombination in E. Coli is the interaction of a RecA nucleoprotein filament formed on DNA with a second DNA molecule. The filament-DNA interaction governs the search for sequence homology, sequence recognition, and eventually the exchange of homologous strands. Here, we employ a dual molecule assay to study the mechanics of this two-molecule interaction.In our experiments, a RecA filament is manipulated in 3D by dual-bead optical tweezers, and brought into contact with a second dsDNA molecule tethered in a magnetic-tweezers configuration. The strength of the interaction is studied using a force-spectroscopy technique with the magnetic bead as the sensitive force probe (resolution 10 fN/sqrt(Hz)). We present results on the interaction between supercoiled dsDNA and RecA filaments formed on dsDNA. We find that the strength of interaction depends sensitively on the supercoiling state of the dsDNA with stronger interactions recorded for negatively supercoiled DNA. This observation is interpreted as enhanced binding of DNA in a denatured state to the secondary binding site of the RecA filament. The strong affinity of the RecA filament for denatured dsDNA could lead to a favoring of the search process to regions of denatured dsDNA where base sampling is possible, a feature that is expected to be important in governing a fast and efficient homology search process.

DNA Binding, Coprotease, and Strand Exchange Activities of Mycobacterial RecA Proteins: Implications for Functional Diversity among RecA Nucleoprotein Filaments

One of the fundamental questions concerning homologous recombination is how RecA or its homologues recognize several DNA sequences with high affinity and catalyze all the diverse biological activities. In this study, we show that the extent of single-stranded DNA binding and strand exchange (SE) promoted by mycobacterial RecA proteins with DNA substrates having various degrees of GC content was comparable with that observed for Escherichia coli RecA. However, the rate and extent of SE promoted by these recombinases showed a strong negative correlation with increasing amounts of sequence divergence embedded at random across the length of the donor strand. Conversely, a positive correlation was seen between SE efficiency and the degree of sequence divergence in the recipient duplex DNA. The extent of heteroduplex formation was not significantly affected when both the pairing partners contained various degrees of sequence divergence, although there was a moderate decrease in the case of mycobacterial RecA proteins with substrates containing larger amounts of sequence divergence. Whereas a high GC content had no discernible effect on E. coli RecA coprotease activity, a negative correlation was apparent between mycobacterial RecA proteins and GC content. We further show clear differences in the extent of SE promoted by E. coli and mycobacterial RecA proteins in the presence of a wide range of ATP:ADP ratios. Taken together, our findings disclose the existence of functional diversity among E. coli and mycobacterial RecA nucleoprotein filaments, and the milieu of sequence divergence (i.e., in the donor or recipient) exerts differential effects on heteroduplex formation, which has implications for the emergence of new genetic variants.

Modeling the early stage of DNA sequence recognition within RecA nucleoprotein filaments

Homologous recombination is a fundamental process enabling the repair of double-strand breaks with a high degree of fidelity. In prokaryotes, it is carried out by RecA nucleofilaments formed on single-stranded DNA (ssDNA). These filaments incorporate genomic sequences that are homologous to the ssDNA and exchange the homologous strands. Due to the highly dynamic character of this process and its rapid propagation along the filament, the sequence recognition and strand exchange mechanism remains unknown at the structural level. The recently published structure of the RecA/DNA filament active for recombination (Chen et al., Mechanism of homologous recombination from the RecA-ssDNA/dsDNA structure, Nature 2008, 453, 489) provides a starting point for new exploration of the system. Here, we investigate the possible geometries of association of the early encounter complex between RecA/ssDNA filament and double-stranded DNA (dsDNA). Due to the huge size of the system and its dense packing, we use a reduced representation for protein and DNA together with state-of-the-art molecular modeling methods, including systematic docking and virtual reality simulations. The results indicate that it is possible for the double-stranded DNA to access the RecA-bound ssDNA while initially retaining its Watson–Crick pairing. They emphasize the importance of RecA L2 loop mobility for both recognition and strand exchange.

Mycobacterium tuberculosis UvrD1 and UvrA Proteins Suppress DNA Strand Exchange Promoted by Cognate and Noncognate RecA Proteins

DNA helicases are present in all kingdoms of life and play crucial roles in processes of DNA metabolism such as replication, repair, recombination, and transcription. To date, however, the role of DNA helicases during homologous recombination in mycobacteria remains unknown. In this study, we show that Mycobacterium tuberculosis UvrD1 more efficiently inhibited the strand exchange promoted by its cognate RecA, compared to noncognate Mycobacterium smegmatis or Escherichia coli RecA proteins. The M. tuberculosis UvrD1(Q276R) mutant lacking the helicase and ATPase activities was able to block strand exchange promoted by mycobacterial RecA proteins but not of E. coli RecA. We observed that M. tuberculosis UvrA by itself has no discernible effect on strand exchange promoted by E. coli RecA but impedes the reaction catalyzed by the mycobacterial RecA proteins. Our data also show that M. tuberculosis UvrA and UvrD1 can act together to inhibit strand exchange promoted by mycobacterial RecA proteins. Taken together, these findings raise the possibility that UvrD1 and UvrA might act together in vivo to counter the deleterious effects of RecA nucleoprotein filaments and/or facilitate the dissolution of recombination intermediates. Finally, we provide direct experimental evidence for a physical interaction between M. tuberculosis UvrD1 and RecA on one hand and RecA and UvrA on the other hand. These observations are consistent with a molecular mechanism, whereby M. tuberculosis UvrA and UvrD1, acting together, block DNA strand exchange promoted by cognate and noncognate RecA proteins.

Atomic Force Microscopy Shows that Chi Sequences and SSB Proteins Prevent DNA Reannealing Behind the Translocating AddAB Helicase-Nuclease

Recombinational repair of DNA breaks requires processing of a DNA end to a 3′-ssDNA overhang. In B.subtilis, this task is done by the helicase-nuclease AddAB which generates ssDNA overhangs terminated at a recombination hotspot (Chi) sequence. This is a substrate for the formation of a RecA nucleoprotein filament that searches for a homologous donor molecule and catalyses DNA strand exchange to promote repair. In this study, we have used AFM to visualize the products of reactions including AddAB and double-stranded DNA molecules. AFM images consistently showed a remaining population of apparently unprocessed dsDNA molecules. The fraction of unprocessed molecules dropped upon addition of increasing concentrations of SSB protein or larger amounts of AddAB protein. Moreover, a larger fraction of DNA molecules were processed to ssDNA when DNA substrates contained the regulatory Chi sequence. Our results are consistent with a model in which the DNA strands reanneal behind the translocating AddAB enzyme. This effect is suppressed by destabilizing the interaction between DNA strands via binding of SSB or multiple AddAB motors, or by the interaction between AddAB and Chi during translocation.

Using Real-Time, Single-Molecule Experiments To Monitor RecA-Mediated Pairing and Strand Exchange Reactions in Various Nucleotide States

AbstractWe have developed tethered particle motion (TPM) experiments to investigate the details of E. coli RecA-mediated pairing and strand exchange at the single-molecule level. Experiments with beads labeled on either the invasion strand or the outgoing strand, both showed that DNA pairing and strand exchange occurs successfully in ATP or its non-hydrolyzable analog, ATPγS. RecA-mediated strand exchange efficiency is similar for ATP and ATPγS, suggesting that ATP hydrolysis by RecA is not required to complete strand exchange in these substrates. In addition, the RecA-mediate strand exchange is shown to progress from 5’end to 3’ end within a small synapsis segment while the exchanged strand is temporally bound to heteroduplex DNA and eventually released without ATP-hydrolysis. One the other hand, experiments with beads labeled on the invasion strand either homologous or heterogeneous to surface anchored duplex DNA showed that there were a lot of transient synapse events existing only from ms to seconds long. The lifetime of these synaptic related to synapse states failing to pass on strand exchange states represents the stability of synaptic intermediate which is correspondence with the strand exchange efficiency. The lifetime values of synaptic state are 1.960.10s and 2.740.38s for 229mer and 427mer single strand DNA respectively. Moreover, the initial Brownian motion amplitude comes from the length contribution of the RecA nucleoprotein filament and interacted surface anchored duplex DNA. A sum of two-dice model is adapted to simulate the initial pairing event and shows that synaptic-complex formation follows a random collision initial step without invasion end preference for unsuccessful strand exchange events. One the contrary, 5’ end invasion preference is required to drive synaptic state efficiently pass on the strand exchange process.

An SOS Inhibitor that Binds to Free RecA Protein: The PsiB Protein

The process of bacterial conjugation involves the transfer of a conjugative plasmid as a single strand. The potentially deleterious SOS response, which is normally triggered by the appearance of single-stranded DNA, is suppressed in the recipient cell by a conjugative plasmid system centered on the product of the psiB gene. The F plasmid PsiB protein inhibits all activities of the RecA protein, including DNA binding, DNA strand exchange, and LexA protein cleavage. The proteins known to negatively regulate recombinases, such as RecA or Rad51, generally work at the level of dismantling the nucleoprotein filament. However, PsiB binds to RecA protein that is free in solution. The RecA-PsiB complex impedes formation of RecA nucleoprotein filaments on DNA.

The active form of DNA polymerase V is UmuD′2C–RecA–ATP

DNA-damage-induced SOS mutations arise when Escherichia coli DNA polymerase (pol) V, activated by a RecA nucleoprotein filament (RecA*), catalyses translesion DNA synthesis. Here we address two longstanding enigmatic aspects of SOS mutagenesis, the molecular composition of mutagenically active pol V and the role of RecA*. We show that RecA* transfers a single RecA-ATP stoichiometrically from its DNA 3'-end to free pol V (UmuD'(2)C) to form an active mutasome (pol V Mut) with the composition UmuD'(2)C-RecA-ATP. Pol V Mut catalyses TLS in the absence of RecA* and deactivates rapidly upon dissociation from DNA. Deactivation occurs more slowly in the absence of DNA synthesis, while retaining RecA-ATP in the complex. Reactivation of pol V Mut is triggered by replacement of RecA-ATP from RecA*. Thus, the principal role of RecA* in SOS mutagenesis is to transfer RecA-ATP to pol V, and thus generate active mutasomal complex for translesion synthesis.

Influence of Polyelectrolyte on DNA–RecA Nucleoprotein Filaments: Poly‐L‐Lysine Used as a Model

RecA of Escherichia coli and its active nucleoprotein filaments with DNA are important for the genomic integrity and the genetic diversity. The formation of the DNA-RecA nucleoprotein filaments is a complex multiple-step process and can be affected by many factors. In this work, the effects of poly-L-lysine (PLL) on the DNA-RecA nucleoprotein filaments are investigated in vitro by agarose gel electrophoresis and atomic force microscopy (AFM). The observed morphologies vary with the concentration, the length, and the addition order of PLL. These distinctions provide information for the conformation change of DNA and the binding sites of RecA protein in the formation process of nucleoprotein filaments. Besides that, the comparison displays the effect of cationic polyelectrolyte on both inhibition and destabilization of the DNA-RecA nucleoprotein filaments. The current study provides valuable insights for pharmacologic studies and gene delivery.

Reconstitution of initial steps of dsDNA break repair by the RecF pathway of E. coli

The RecF pathway of Escherichia coli is important for recombinational repair of DNA breaks and gaps. Here ;we reconstitute in vitro a seven-protein reaction that recapitulates early steps of dsDNA break repair using purified RecA, RecF, RecO, RecR, RecQ, RecJ, and SSB proteins, components of the RecF system. Their combined action results in processing of linear dsDNA and its homologous pairing with supercoiled DNA. RecA, RecO, RecR, and RecJ are essential for joint molecule formation, whereas SSB and RecF are stimulatory. This reconstituted system reveals an unexpected essential function for RecJ exonuclease: the capability to resect duplex DNA. RecQ helicase stimulates this processing, but also disrupts joint molecules. RecO and RecR have two indispensable functions: They mediate exchange of RecA for SSB to form the RecA nucleoprotein filament, and act with RecF to load RecA onto the SSB-ssDNA complex at processed ssDNA-dsDNA junctions. The RecF pathway has many parallels with recombinational repair in eukaryotes.

Defective Dissociation of a “Slow” RecA Mutant Protein Imparts an Escherichia coli Growth Defect

The RecA and some related proteins possess a simple motif, called (KR)X(KR), that (in RecA) consists of two lysine residues at positions 248 and 250 at the subunit-subunit interface. This study and previous work implicate this RecA motif in the following: (a) catalyzing ATP hydrolysis in trans,(b) coordinating the ATP hydrolytic cycles of adjacent subunits, (c) governing the rate of ATP hydrolysis, and (d) coupling the ATP hydrolysis to work (in this case DNA strand exchange). The conservative K250R mutation leaves RecA nucleoprotein filament formation largely intact. However, ATP hydrolysis is slowed to less than 15% of the wild-type rate. DNA strand exchange is also slowed commensurate with the rate of ATP hydrolysis. The results reinforce the idea of a tight coupling between ATP hydrolysis and DNA strand exchange. When a plasmid-borne RecA K250R protein is expressed in a cell otherwise lacking RecA protein, the growth of the cells is severely curtailed. The slow growth defect is alleviated in cells lacking RecFOR function, suggesting that the defect reflects loading of RecA at stalled replication forks. Suppressors occur as recA gene alterations, and their properties indicate that limited dissociation by RecA K250R confers the slow growth phenotype. Overall, the results suggest that recombinational DNA repair is a common occurrence in cells. RecA protein plays a sufficiently intimate role in the bacterial cell cycle that its properties can limit the growth rate of a bacterial culture.