RecA Molecules Research Articles

The Interaction of RecA With Both CheA and CheW Is Required for Chemotaxis.

Salmonella enterica is the most frequently reported cause of foodborne illness. As in other microorganisms, chemotaxis affords key physiological benefits, including enhanced access to growth substrates, but also plays an important role in infection and disease. Chemoreceptor signaling core complexes, consisting of CheA, CheW and methyl-accepting chemotaxis proteins (MCPs), modulate the switching of bacterial flagella rotation that drives cell motility. These complexes, through the formation of heterohexameric rings composed of CheA and CheW, form large clusters at the cell poles. RecA plays a key role in polar cluster formation, impairing the assembly when the SOS response is activated. In this study, we determined that RecA protein interacts with both CheW and CheA. The binding of these proteins to RecA is needed for wild-type polar cluster formation. In silico models showed that one RecA molecule, attached to one signaling unit, fits within a CheA-CheW ring without interfering with the complex formation or array assembly. Activation of the SOS response is followed by an increase in RecA, which rises up the number of signaling complexes associated with this protein. This suggests the presence of allosteric inhibition in the CheA-CheW interaction and thus of heterohexameric ring formation, impairing the array assembly. STED imaging demonstrated that all core unit components (CheA, CheW, and MPCs) have the same subcellular location as RecA. Activation of the SOS response promotes the RecA distribution along the cell instead of being at the cell poles. CheA- and CheW- RecA interactions are also crucial for chemotaxis, which is maintained when the SOS response is induced and the signaling units are dispersed. Our results provide new molecular-level insights into the function of RecA in chemoreceptor clustering and chemotaxis determining that the impaired chemoreceptor clustering not only inhibits swarming but also modulates chemotaxis in SOS-induced cells, thereby modifying bacterial motility in the presence of DNA-damaging compounds, such as antibiotics.

Structure and interactions of RecA: plasticity revealed by molecular dynamics simulations

Eleven independent simulations, each involving three consecutive molecules in the RecA filament, carried out on the protein from Mycobacterium tuberculosis, Mycobacterium smegmatis and Escherichia coli and their Adenosine triphosphate (ATP) complexes, provide valuable information which is complementary to that obtained from crystal structures, in addition to confirming the robust common structural framework within which RecA molecules from different eubacteria function. Functionally important loops, which are largely disordered in crystal structures, appear to adopt in each simulation subsets of conformations from larger ensembles. The simulations indicate the possibility of additional interactions involving the P-loop which remains largely invariant. The phosphate tail of the ATP is firmly anchored on the loop while the nucleoside moiety exhibits substantial structural variability. The most important consequence of ATP binding is the movement of the ‘switch’ residue. The relevant simulations indicate the feasibility of a second nucleotide binding site, but the pathway between adjacent molecules in the filament involving the two nucleotide binding sites appears to be possible only in the mycobacterial proteins.

Protein Adsorption on Hybrids of Thermoresponsive Polymers and Single-Walled Carbon Nanotubes

Poly(N-isopropylacrylamide) (PNIPAAm) is one of the most popular thermoresponsive polymers. Adsorption of RecA proteins onto hybrids of PNIPAAm and single-walled carbon nanotubes (SWNTs) was observed in the presence and absence of DNA molecules. Although RecA molecules were adsorbed efficiently onto the hybrid surfaces at 37°C, even in the absence of DNA molecules, the adsorption of RecA was inhibited at 4°C. These results suggest that the thermoresponsive functions of PNIPAAm were effective, even on the SWNT surfaces, which supports the possibility of developing nanobiodevices using PNIPAAm-SWNT hybrids. However, although RecA is a DNA binding protein, there was no significant difference in the adsorption of RecA onto PNIPAAm-SWNT surfaces with and without DNA molecules. This study provides fundamental information for potential biological applications of PNIPAAm-SWNT hybrids.

Biomolecular recognition ability of RecA proteins for DNA on single-walled carbon nanotubes

We examined the biomolecular recognition ability of RecA proteins using single-walled carbon nanotubes (SWNTs) wrapped with a single-stranded DNA (ssDNA) molecule as a mimic for the usual ssDNA molecules. The ssDNA-SWNT hybrids showed larger diameters compared to those of the usual ssDNA molecules. As a result, RecA molecules bound to the ssDNA-SWNTs, as observed using atomic force microscopy and agarose gel electrophoresis. On the other hand, when carboxymethylcellulose (CMC) was used rather than ssDNA, the RecA molecules did not bind to the CMC-SWNT hybrids. Our results indicate that RecA molecules recognize ssDNA on SWNT surfaces as DNA molecules through their biomolecular recognition ability.

RecA bundles mediate homology pairing between distant sisters during DNA break repair

DNA double-strand break (DSB) repair by homologous recombination (HR) has evolved to maintain genetic integrity in all organisms1. Although many reactions that occur during HR are known1-3, it is unclear where, when and how they occur in cells is lacking. Here, by using conventional and super-resolution microscopy we describe the progression of DSB repair in live Escherichia coli. Specifically, we investigate whether HR can occur efficiently between distant sister loci that have segregated to opposite halves of an E. coli cell. We show that a site-specific DSB in one sister can be repaired efficiently using distant sister homology. After RecBCD processing of the DSB, RecA is recruited to the cut locus, where it nucleates into a bundle that contains many more RecA molecules than can associate with the two ssDNA regions that form at the DSB. Mature bundles extend along the cell long axis in the space between the bulk nucleoid and the inner membrane. Bundle formation is followed by pairing in which the two ends of the cut locus relocate at the periphery of the nucleoid and together move rapidly towards the homology of the uncut sister. After sister locus pairing, RecA bundles disassemble and proteins that act late in HR are recruited to give viable recombinants 1-2 generation time equivalents after formation of the initial DSB. Mutated RecA proteins that do not form bundles are defective in sister pairing and in DSB-induced repair. The work reveals an unanticipated role of RecA bundles in channeling the movement of the DNA DSB ends, thereby facilitating the long-range homology search that occurs before the strand invasion and transfer reactions.

Functionally important movements in RecA molecules and filaments: studies involving mutation and environmental changes. Corrigendum

Functionally important movements in RecA molecules and filaments: studies involving mutation and environmental changes. Corrigendum

Modulation of UvrD Helicase Activity by Covalent DNA-Protein Cross-links

UvrD (DNA helicase II) has been implicated in DNA replication, DNA recombination, nucleotide excision repair, and methyl-directed mismatch repair. The enzymatic function of UvrD is to translocate along a DNA strand in a 3' to 5' direction and unwind duplex DNA utilizing a DNA-dependent ATPase activity. In addition, UvrD interacts with many other proteins involved in the above processes and is hypothesized to facilitate protein turnover, thus promoting further DNA processing. Although UvrD interactions with proteins bound to DNA have significant biological implications, the effects of covalent DNA-protein cross-links on UvrD helicase activity have not been characterized. Herein, we demonstrate that UvrD-catalyzed strand separation was inhibited on a DNA strand to which a 16-kDa protein was covalently bound. Our sequestration studies suggest that the inhibition of UvrD activity is most likely due to a translocation block and not helicase sequestration on the cross-link-containing DNA substrate. In contrast, no inhibition of UvrD-catalyzed strand separation was apparent when the protein was linked to the complementary strand. The latter result is surprising given the earlier observations that the DNA in this covalent complex is severely bent ( approximately 70 degrees ), with both DNA strands making multiple contacts with the cross-linked protein. In addition, UvrD was shown to be required for replication of plasmid DNAs containing covalent DNA-protein complexes. Combined, these data suggest a critical role for UvrD in the processing of DNA-protein cross-links.

Investigating the Nucleation and Extension Rates of E.coli and Deinococcus RecA Along Duplex DNA

RecA is a protein which promotes the exchange between two homologous DNA molecules in homologous recombination process. When individual RecA molecules assemble on DNA, the DNA is stretched and underwound to form a nucleoprotein filament with its rigidity and end-to-end length increased. We have developed single-molecule tethered particle motion (TPM) experiments to study the assembly dynamics of RecA proteins on individual duplex DNA molecules. The TPM method is capable of measuring the changes in DNA length by observing the bead's Brownian motion, thus allowing us to monitor RecA nucleation and extension in real-time. Using much shorter DNA (a few hundreds basepairs), TPM experiments offer improved sensitivity, since the DNA length change can be readily detected as soon as a few RecA bounded to duplex DNA molecules. Our experiments indicated a faster nucleation rate compared to the previous reports (Galletto et al., 2006). Moreover, we have compared the nucleation and extension rates of E. coli RecA with the RecA from Deinococcus radiodurans, UV-resistant bacteria, under different nucleotide states, ATP and ATPγS. Deinococcus radiodurans RecA are found to nucleate faster (∼1.6×10-2 bp-1min-1) but extend slower (∼0.3-1.5 RecA/sec) under ATP.This difference reflects the physiological role of Dr. RecA when extensive UV-damaged DNA molecules are present.

Functionally important movements in RecA molecules and filaments: studies involving mutation and environmental changes

The crystal structures of mutants of Mycobacterium smegmatis RecA (MsRecA) involving changes of Gln196 from glutamine to alanine, asparagine and glutamic acid, wild-type MsRecA and several of their nucleotide complexes have been determined using mostly low-temperature and partly room-temperature X-ray data. At both temperatures, nucleotide binding results in a movement of Gln196 towards the bound nucleotide in the wild-type protein. This movement is abolished in the mutants, thus establishing the structural basis for the triggering action of the residue in terms of the size, shape and the chemical nature of the side chain. The 19 crystal structures reported here, together with 11 previously reported MsRecA structures, provide further elaboration of the relation between the pitch of the ;inactive' RecA filament, the orientation of the C-terminal domain with respect to the main domain and the location of the switch residue. The low-temperature structures define one extreme of the range of positions the C-terminal domain can occupy. The movement of the C-terminal domain is correlated with those of the LexA-binding loop and the loop that connects the main and the N-terminal domains. These elements of molecular plasticity are made use of in the transition to the ;active' filament, as evidenced by the recently reported structures of RecA-DNA complexes. The available structures of RecA resulting from X-ray and electron-microscopic studies appear to represent different stages in the trajectory of the allosteric transformations of the RecA filament. The work reported here contributes to the description of the early stages of this trajectory and provides insight into structures relevant to the later stages.

Expedient placement of two fluorescent dyes for investigating dynamic DNA protein interactions in real time.

Many questions in molecular and cellular biology can be reduced to questions about 'who talks to whom, when and how frequently'. Here, we review approaches we have used with single-pair fluorescence resonance energy transfer (spFRET) to follow the motions between two well-placed fluorescent probes to ask similar questions. We describe two systems. We have used a nucleosomal system in which the naked DNA molecule has the acceptor and donor dyes too far apart for FRET to occur whereas the dyes are close enough in the reconstituted nucleosome for FRET. As these individual nucleosomes were tethered on a surface, we could follow dynamics in the repositioning of these two dyes, inferring that nucleosomes stochastically and reversibly open and close. These results imply that most of the DNA on the nucleosome can be sporadically accessible to regulatory proteins and proteins that track the DNA double helix. In the case of following the binding of recombination protein RecA to double-stranded DNA (dsDNA) and the RecA filament displacement by DNA helicase motor PcrA, the dsDNA template is prepared with the two dyes close enough to each other to generate high FRET. Binding of the RecA molecules to form a filament lengthens the dsDNA molecule 1.5-fold and reduces the FRET accordingly. Once added, DNA motor protein helicase PcrA can displace the RecA filament with concomitant return of the DNA molecule to its original B-form and high FRET state. Thus, appropriately placed fluorescent dyes can be used to monitor conformational changes occurring in DNA and or proteins and provide increased sensitivity for investigating dynamic DNA-protein interactions in real time.

Geometry of the DNA strands within the RecA nucleofilament: role in homologous recombination.

Homologous recombination consists of exchanging DNA strands of identical or almost identical sequence. This process is important for both DNA repair and DNA segregation. In prokaryotes, it involves the formation of long helical filaments of the RecA protein on DNA. These filaments incorporate double-stranded DNA from the cell's genetic material, recognize sequence homology and promote strand exchange between the two DNA segments. DNA processing by these nucleofilaments is characterized by large amplitude deformations of the double helix, which is stretched by 50% and unwound by 40% with respect to B-DNA. In this article, information concerning the structure and interactions of the RecA, DNA and ATP molecules involved in DNA strand exchange is gathered and analyzed to present a view of their possible arrangement within the filament, their behavior during strand exchange and during ATP hydrolysis, the mechanism of RecA-promoted DNA deformation and the role of DNA deformation in the process of homologous recombination. In particular, the unusual characteristics of DNA within the RecA filament are compared to the DNA deformations locally induced by architectural proteins which bind in the DNA minor groove. The possible role and location of two flexible loops of RecA are discussed.

Two distinct modes of RecA action are required for DNA polymerase V-catalyzed translesion synthesis.

SOS mutagenesis in Escherichia coli requires DNA polymerase V (pol V) and RecA protein to copy damaged DNA templates. Here we show that two distinct biochemical modes for RecA protein are necessary for pol V-catalyzed translesion synthesis. One RecA mode is characterized by a strong stimulation in nucleotide incorporation either directly opposite a lesion or at undamaged template sites, but by the absence of lesion bypass. A separate RecA mode is necessary for translesion synthesis. The RecA1730 mutant protein, which was identified on the basis of its inability to promote pol V (UmuD'(2)C)-dependent UV-mutagenesis, appears proficient for the first mode of RecA action but is deficient in the second mode. Data are presented suggesting that the two RecA modes are "nonfilamentous". That is, contrary to current models for SOS mutagenesis, formation of a RecA nucleoprotein filament may not be required for copying damaged DNA templates. Instead, SOS mutagenesis occurs when pol V interacts with two RecA molecules, first at a 3' primer end, upstream of a template lesion, where RecA mode 1 stimulates pol V activity, and subsequently at a site immediately downstream of the lesion, where RecA mode 2 cocatalyzes lesion bypass. We posit that in vivo assembly of a RecA nucleoprotein filament may be required principally to target pol V to a site of DNA damage and to stabilize the pol V-RecA interaction at the lesion. However, it is only a RecA molecule located at the 3' filament tip, proximal to a damaged template base, that is directly responsible for translesion synthesis.

Recombinogenic activity of chimeric recA genes (Pseudomonas aeruginosa/Escherichia coli): a search for RecA protein regions responsible for this activity.

In the background of weak, if any, constitutive SOS function, RecA from Pseudomonas aeruginosa (RecAPa) shows a higher frequency of recombination exchange (FRE) per DNA unit length as compared to RecA from Escherichia coli (RecAEc). To understand the molecular basis for this observation and to determine which regions of the RecAPa polypeptide are responsible for this unusual activity, we analyzed recAX chimeras between the recAEc and recAPa genes. We chose 31 previously described recombination- and repair-proficient recAX hybrids and determined their FRE calculated from linkage frequency data and constitutive SOS function expression as measured by using the lacZ gene under control of an SOS-regulated promoter. Relative to recAEc, the FRE of recAPa was 6.5 times greater; the relative alterations of FRE for recAX genes varied from approximately 0.6 to 9.0. No quantitative correlation between the FRE increase and constitutive SOS function was observed. Single ([L29M] or [I102D]), double ([G136N, V142I]), and multiple substitutions in related pairs of chimeric RecAX proteins significantly altered their relative FRE values. The residue content of three separate regions within the N-terminal and central but not the C-terminal protein domains within the RecA molecule also influenced the FRE values. Critical amino acids in these regions were located close to previously identified sequences that comprise the two surfaces for subunit interactions in the RecA polymer. We suggest that the intensity of the interactions between the subunits is a key factor in determining the FRE promoted by RecA in vivo.

Single-molecule studies of DNA and DNA–protein interactions

Two experiments are described that examine the properties of a single DNA molecule. The experimental system is based on optical tweezers and on a protocol that allows to attach one end of the DNA molecule to the bottom of the sample and the other to a bead of micron size. First, the reaction of the RecA protein with DNA is monitored in a particular DNA molecule. This is made possible by the fact that the DNA is locally extended by a factor of 1.5 whenever a RecA molecule binds. Second, we monitor the dynamics of a DNA molecule as it relaxes from a fully stretched state to the coil configuration.

RecA protein has extremely high cooperativity for substrate in its ATPase activity.

The single-stranded DNA-dependent ATPase activity of Escherichia coli RecA protein, especially its cooperativity for ATP, was investigated. To measure the ATPase activity in detail, the methods and reaction conditions for the ATPase assay were reexamined. Under conditions where RecA protein always showed a maximal rate of ATP hydrolysis, its poly(dT)-dependent ATPase activity was measured. At 25 degrees C, increasing the concentration of RecA protein from 0.3 to 1.0 microM increased the turnover number (kcat) from 0.16 to 0.19 s-1 and the Hill coefficient (nH) for ATP from 9.3 to 11.6. At 0.5 microM RecA protein, increasing the temperature from 25 to 37 degrees C increased kcat from 0.18 to 0.35 s-1 but decreased nH from 9.8 to 6.6. Interestingly, the ATPase activity of RecA protein measured in this study showed much higher cooperativity for ATP than those reported to date. Furthermore, the nH value of 11.6 for ATP obtained here was the highest of any ATPase reported so far. These results suggest that the binding of an ATP molecule to a RecA molecule within a nucleoprotein helical filament causes structural change of many other neighboring RecA molecules. This implies that ATP binding induces structural change of the whole nucleoprotein helical filament. Finally, we demonstrated that analysis of cooperativity is useful for revealing how a protein composed of many subunits functions as a whole.

The identification of the single-stranded DNA-binding domain of the Escherichia coli RecA protein.

To identify the ssDNA-binding domain of Escherichia coli RecA protein, we examined the ssDNA-binding capabilities of synthetic peptides, the sequences of which were derived from the C- and N-termini and from sequences within loops L1 and L2 of the RecA molecule identified from the crystal structure. Synthetic peptides derived from amino acid residues 185-219 of several bacterial RecA proteins, which include loop L2 of RecA, bound to ssDNA in filter-binding assays, whereas three separate synthetic peptides corresponding to single point mutants of E. coli RecA in this region did not. The binding of RecA to ssDNA examined using a gel-shift assay was inhibited by a synthetic peptide derived from this ssDNA-binding region, but not by synthetic peptides derived from amino acid residues 301-329 of the C-terminus or from N-terminal residues 6-39. A peptide corresponding to amino acid positions 152-169 of the RecA molecule and spanning loop L1 and its flanking regions did not bind ssDNA at peptide concentrations up to 250 microM. We have also defined a synthetic 20-amino-acid peptide that comprises amino acid residues 193-212 and includes loop L2 of RecA as the minimum unit that can bind to ssDNA from this region of RecA. Finally, two maltose-binding protein-RecA fusion proteins were made, one containing amino acid residues 185-224 of RecA and the other the last 51 C-terminal residues of RecA (amino acid residues 303-353). In contrast to the C-terminus-derived fusion protein, the fusion protein containing the putative DNA-binding site demonstrated significant binding to single-stranded oligonucleotides in both filter-binding and gel-shift assays. These findings suggest that a portion of the region extending from amino acid residues 193-212 is either part of or the whole ssDNA-binding domain of the RecA protein.

Analysis of the DNA binding site of Escherichia coli RecA protein

To investigate the DNA binding site of RecA protein, we constructed 15 recA mutants having alterations in the regions homologous to the other ssDNA binding proteins. The in vivo analyses showed that the mutational change at Arg 243, Lys 248, Tyr 264, or simultaneously at Lys 6 and Lys 19, or Lys 6 and Lys 23 caused severe defects in the recA functions, while other mutational changes did not. Purified RecA-K6A-K23A (Lys 6 and Lys 23 changed to Ala and Ala, respectively) protein was indistinguishable from the wild-type RecA protein in its binding to DNA. However, the RecA-R243A (Arg 243 changed to Ala) and RecA-Y264A (Tyr 264 changed to Ala) proteins were defective in binding to both ss- and ds-DNA. In self-oligomerization property, RecA-R243A was proficient but RecA-Y264A was deficient, suggesting that the RecA-R243A protein had a defect in DNA binding site and the RecA-Y264A protein was defective in its interaction with the adjacent RecA molecule. The region of residues 243–257 including the Arg 243 is highly homologous to the DNA binding motif in the ssDNA binding proteins, while the eukaryotic RecA homologues have a similar structure at the amino-terminal side proximal to the nucleotide binding core. The region of residues 243–257 would be a part of the DNA binding site. The other parts of this site would be the Tyr 103 and the region of residues 178–183, which were cross-linked to ssDNA. These three regions lie in a line in the crystal structure.

Structure of RecA-DNA complex and mechanism of DNA strand exchange reaction in homologous recombination.

The importance of filament formation of RecA for the DNA strand exchange reaction, both in vivo and in vitro, is established. RecA forms a very long and relatively stiff filament by binding around DNA with high cooperativity. The monomer units are assembled in the filament in a head-to-tail arrangement in a helical manner, similar to the organization of RecA molecules found in the crystal of pure RecA or including ADP. This filament of RecA, containing a DNA molecule in its interior, can bind another DNA molecule and yet a third one in the presence of cofactor (ATP or its analogs). Each filament may have three DNA binding sites, each able to bind one DNA strand of either ss or ds DNA. According to linear dichroism and fluorescence spectroscopies, the DNA molecules in the RecA filament are well organized with a well defined but modified structure. This organization and modification of DNA by RecA probably has the purpose of facilitating Watson-Crick base-pair recognition and strand exchange reaction. RecA is thus actively involved in the reaction. The phosphoribose backbone of DNA follows the RecA helix and the DNA is stretched 50% and unwound. The nucleobases are destacked but still firmly oriented almost perpendicular to the axis for the first DNA and are immobile even in the case of ssDNA. Even in the second DNA the motion of DNA bases is very restricted although their orientation appears to be less perpendicular. All DNA strands in the complex show some sequence dependent interaction with each other. It could be a non-conventional base-base pairing, that could provide a mechanism of search for homologous DNA. In order to understand how RecA organizes DNA, a 3-D model building of the RecA-DNA complex is in progress undertaken based on the crystal structure of RecA and results obtained using chemical interference and protein engineering techniques. A characterization of structure of the second DNA and the mode of DNA-DNA interaction may further clarify the reaction mechanism of strand exchange.

Accessibility to modification of histidine residues of RecA protein upon DNA and cofactor binding.

The potential role of histidine residues of RecA protein in binding DNA has been investigated by monitoring their accessibility to diethylpyrocarbonate. In the absence of both DNA and cofactor, only one of two histidine residues is modified by the reagent, indicating that the other residue is buried. However, both histidine residues become accessible after addition of cofactor analog adenosine 5'-O-(3-thiotriphosphate) (ATP[S]) indicating a change in the organization of the RecA filament and/or a change in the conformation of protein. The diethylpyrocarbonate-modified RecA is found to be able to polymerize just as the unmodified protein. The binding of double-stranded DNA, in the presence of ATP[S], reduces the reactivity of both histidine residues to diethylpyrocarbonate. The binding of single-stranded DNA (with ATP[S]) has a similar, though smaller, protective effect. However, no significant dissociation of either of the complexes as a result of the modification was observed and a RecA molecule which had been modified in the absence of DNA could still bind DNA. A protection of the histidine residues is also effected by high salt concentration which promotes, just as DNA binding, ATPase and coprotease activity in RecA. The protection of histidine residues to diethylpyrocarbonate upon DNA binding probably relates to a conformational change of RecA and may not be any direct effect of shielding by the DNA. Nonetheless, the domains including the histidine residues could be centers of allosteric effects and are concluded to be close to the DNA binding site.

Use of ion exchange chromatography for the study of RecA-DNA interaction

In vitro binding or RecA protein to double-stranded DNA (dsDNA) was studied using ion-exchange liquid chromatography. The method allowed quantification of both free DNA and free protein. The results unambiguously showed a binding stoichiometry or 3 base pairs per RecA monomer. The binding exhibited cooperativity, and the stoichiometry suggested that RecA does not form complexes with two molecules or dsDNA. More than 90% of RecA molecules in the sample were active for DNA binding.