RecA Activity Research Articles

The RdgC protein of Escherichia coli binds DNA and counters a toxic effect of RecFOR in strains lacking the replication restart protein PriA.

PriA protein provides a means to load the DnaB replicative helicase at DNA replication fork and D loop structures, and is therefore a key factor in the rescue of stalled or broken forks and subsequent replication restart. We show that the nucleoid-associated RdgC protein binds non-specifically to single-stranded (ss) DNA and double-stranded DNA. It is also essential for growth of a strain lacking PriA, indicating that it might affect replication fork progression or fork rescue. dnaC suppressors of priA overcome this inviability, especially when RecF, RecO or RecR is inactivated, indicating that RdgC avoids or counters a toxic effect of these proteins. Mutations modifying ssDNA-binding (SSB) protein also negate this toxic effect, suggesting that the toxicity reflects inappropriate loading of RecA on SSB-coated ssDNA, leading to excessive or untimely RecA activity. We suggest that binding of RdgC to DNA limits RecA loading, avoiding problems at replication forks that would otherwise require PriA to promote replication restart. Mutations in RNA polymerase also reduce the toxic effect of RecFOR, providing a further link between DNA replication, transcription and repair.

ATP-Mediated Conformational Changes in the RecA Filament

The crystal structure of the E. coli RecA protein was solved more than 10 years ago, but it has provided limited insight into the mechanism of homologous genetic recombination. Using electron microscopy, we have reconstructed five different states of RecA-DNA filaments. The C-terminal lobe of the RecA protein is modulated by the state of the distantly bound nucleotide, and this allosteric coupling can explain how mutations and truncations of this C-terminal lobe enhance RecA's activity. A model generated from these reconstructions shows that the nucleotide binding core is substantially rotated from its position in the RecA crystal filament, resulting in ATP binding between subunits. This simple rotation can explain the large cooperativity in ATP hydrolysis observed for RecA-DNA filaments.

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.

Replication restart in UV-irradiated Escherichia coli involving pols II, III, V, PriA, RecA and RecFOR proteins.

In Escherichia coli, UV-irradiated cells resume DNA synthesis after a transient inhibition by a process called replication restart. To elucidate the role of several key proteins involved in this process, we have analysed the time dependence of replication restart in strains carrying a combination of mutations in lexA, recA, polB (pol II), umuDC (pol V), priA, dnaC, recF, recO or recR. We find that both pol II and the origin-independent primosome-assembling function of PriA are essential for the immediate recovery of DNA synthesis after UV irradiation. In their absence, translesion replication or 'replication readthrough' occurs approximately 50 min after UV and is pol V-dependent. In a wild-type, lexA+ background, mutations in recF, recO or recR block both pathways. Similar results were obtained with a lexA(Def) recF strain. However, lexA(Def) recO or lexA(Def) recR strains, although unable to facilitate PriA-pol II-dependent restart, were able to perform pol V-dependent readthrough. The defects in restart attributed to mutations in recF, recO or recR were suppressed in a recA730 lexA(Def) strain expressing constitutively activated RecA (RecA*). Our data suggest that in a wild-type background, RecF, O and R are important for the induction of the SOS response and the formation of RecA*-dependent recombination intermediates necessary for PriA/Pol II-dependent replication restart. In con-trast, only RecF is required for the activation of RecA that leads to the formation of pol V (UmuD'2C) and facilitates replication readthrough.

Characterization of RecA424 and RecA670 proteins from Deinococcus radiodurans.

RecA protein is considered to be the most important participant in the radiation resistance of Deinococcus radiodurans. However, it is still unclear how RecA contributes to the resistance. In this study, we identified a new recA mutation (recA424) in the DNA-repair deficient mutant strain KI696, the phenotype of which is remarkably different from mutant strain rec30 carrying recA670. The properties of the gene products from the recA mutants were compared. recA424 could not complement the deficiency in Escherichia coli RecA, as found for recA670. In vitro, neither RecA424 nor RecA670 could promote DNA strand exchange under conditions in which wild-type RecA promoted the reaction, indicating that both RecA424 and Rec670 are defective in recombination activity. RecA424 promoted the autocleavage reaction of LexA in vitro, whereas RecA670 did not. The intracellular LexA level in KI696 was decreased following gamma-irradiation. However, the LexA level in strain rec30 was constant irrespective of irradiation. These results indicate that RecA424 retains co-protease activity, whereas RecA670 does not. While strain rec30 is extremely radiation sensitive, strain KI696 is only slightly sensitive. Together, these observations suggest that the co-protease activity rather than the recombination activity of RecA contributes to radiation resistance in D. radiodurans.

Design and Evaluation of a Tryptophanless RecA Protein with Wild Type Activity

The C-terminal domain of the Escherichia coli RecA protein contains two tryptophan residues whose native fluorescence emission provides an interfering background signal when other fluorophores such as 1,N6-ethenoadenine, 2-aminopurine and other tryptophan residues are used to probe the protein's activities. Replacement of the wild type tryptophans with nonfluorescent residues is not trivial because one tryptophan is highly conserved and the C-terminal domain functions in both DNA binding as well as interfilament protein–protein contact. We undertook the task of creating a tryptophanless RecA protein with WT RecA activity by selecting suitable amino acid replacements for Trp290 and Trp308. Mutant proteins were screened in vivo using assays of SOS induction and cell survival following UV irradiation. Based on its activity in these assays, the W290H-W308F W-less RecA was purified for in vitro characterization and functioned like WT RecA in DNA-dependent ATPase and DNA strand exchange assays. Spectrofluorometry indicates that the W290H-W308F RecA protein generates no significant emission when excited with 295-nm light. Based on its ability to function as wild type protein in vivo and in vitro, this dark RecA protein will be useful for future fluorescence experiments.

Physical interactions between DinI and RecA nucleoprotein filament for the regulation of SOS mutagenesis.

The Escherichia coli dinI gene is one of the LexA-regulated genes, which are induced upon DNA damage. Its overexpression conferred severe UV sensitivity on wild-type cells and resulted in the inhibition of LexA and UmuD processing, reactions that are normally dependent on activated RecA in a complex with single-stranded (ss)DNA. Here, we study the mechanism by which DinI inhibits the activities of RecA. While DinI neither binds to ssDNA nor prevents the formation of RecA nucleoprotein filament, it binds to active RecA filament, thereby inhibiting its coprotease activity but not the ATPase activity. Furthermore, even under in vitro conditions where UmuD cleavage dependent on RecA-ssDNA-adeno sine-5'-(3-thiotriphosphate) is blocked in the presence of DinI, LexA is cleaved normally. This result, taken together with electron microscopy observations and linear dichroism measurements, indicates that the ternary complex remains intact in the presence of DinI, and that the affinity to the RecA filament decreases in the order LexA, DinI and UmuD. DinI is thus suited to modulating UmuD processing so as to limit SOS mutagenesis.

Real time fluorescence analysis of the RecA filament: implications of base pair fluidity in repeat realignment

During recombination, when Escherichia coli RecA mediates annealing across DNA repeats, Watson–Crick chemistry can only specify the complementarity of pairing, but not the most optimal frame of alignment. We describe that although stochastic alignments across poly(dA) and poly(dT) can lead to sub-optimally annealed duplexes containing ssDNA gaps/overhangs, the same are realigned into an optimal frame by a putative motor activity of RecA [Sen et al. (2000) Biochemistry 39, 10196–10206]. In the present study, we analyze the nature of realignment intermediates in real time, by employing a fluorescent probe, 2-aminopurine (2AP), which can not only report the status of RecA on the unstacked duplex, but also the fluidity of bases in such a filament. Although known to display a lower affinity for duplex DNA, RecA seems to remain functionally associated with these sub-optimally aligned repeat duplexes, until the realignment approaches completion. Moreover, a comparison of 2AP fluorescence in repeat versus mixed sequences indicates that bases in a RecA repetitive DNA filament exhibit higher degrees of freedom that might mediate a ‘non-planar hydrogen bonding cross talk’ across the bases on either strand. We discuss a model to explain the mechanistic basis of realignment and its implications in signaling the end of homology maximization, which triggers RecA fall off.

A model for the abrogation of the SOS response by an SOS protein: a negatively charged helix in DinI mimics DNA in its interaction with RecA.

DinI is a recently described negative regulator of the SOS response in Escherichia coli. Here we show that it physically interacts with RecA and prevents the binding of single-stranded DNA to RecA, which is required for the activation of the latter. DinI also displaces ssDNA from a stable RecA-DNA cofilament, thus eliminating the SOS signal. In addition, DinI inhibits RecA-mediated homologous DNA pairing, but has no effect on actively proceeding strand exchange. Biochemical data, together with the molecular structure, define the C-terminal alpha-helix in DinI as the active site of the protein. In an unusual example of molecular mimicry, a negatively charged surface on this alpha-helix, by imitating single-stranded DNA, interacts with the loop L2 homologous pairing region of RecA and interferes with the activation of RecA.

RuvABC-dependent double-strand breaks in dnaBts mutants require recA.

Replication fork arrest can cause DNA double-strand breaks (DSBs). These DSBs are caused by the action of the Holliday junction resolvase RuvABC, indicating that they are made by resolution of Holliday junctions formed at blocked forks. In this work, we study the homologous recombination functions required for RuvABC-mediated breakage in cells deficient for the accessory replicative helicase Rep or deficient for the main Escherichia coli replicative helicase DnaB. We show that, in the rep mutant, RuvABC-mediated breakage occurs in the absence of the homologous recombination protein RecA. In contrast, in dnaBts mutants, most of the RuvABC-mediated breakage depends on the presence of RecA, which suggests that RecA participates in the formation of Holliday junctions at forks blocked by the inactivation of DnaB. This action of RecA does not involve the induction of the SOS response and does not require any of the recombination proteins essential for the presynaptic step of homologous recombination, RecBCD, RecF or RecO. Consequently, our observations suggest a new function for RecA at blocked replication forks, and we propose that RecA acts by promoting homologous recombination without the assistance of known presynaptic proteins.

Transcriptional and mutational analyses of the Streptomyces lividans recX gene and its interference with RecA activity.

The role of the 20,922-Da RecX protein and its interference with RecA activity were analyzed in Streptomyces lividans. The recX gene is located 220 bp downstream of recA. Transcriptional analysis by reverse transcriptase PCR demonstrated that recX and recA constitute an operon. While recA was transcribed at a basal level even under noninducing conditions, a recA-recX cotranscript was only detectable after induction of recA following DNA damage. The recA-recX cotranscript was less abundant than the recA transcript alone. The recX gene was inactivated by gene replacement. The resulting mutant had a clearly diminished colony size, but was not impaired in recombination activity, genetic instability, and resistance against UV irradiation. Expression of an extra copy of the S. lividans recA gene under control of the thiostrepton-inducible tipA promoter was lethal to the recX mutant, demonstrating that RecX is required to overcome the toxic effects of recA overexpression. Since inactivation of the recX gene did not influence transcription of recA, the putative function of the RecX protein might be the downregulation of RecA activity by interaction with the RecA protein or filament.

Specific defects in double-stranded DNA unwinding and homologous pairing of a mutant RecA protein

The DNA molecules bound to RecA filaments are extended 1.5-fold relative to B-form DNA. This extended DNA structure may be important in the recognition of homology between single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA). In this study, we show that the K286N mutation specifically impaired the dsDNA unwinding and homologous pairing activities of RecA, without an apparent effect on dsDNA binding itself. In contrast, the R243Q mutation caused defective dsDNA unwinding, due to the defective dsDNA binding of the C-terminal domain of RecA. These results provide new evidence that dsDNA unwinding is essential to homology recognition between ssDNA and dsDNA during homologous pairing.

RecA stimulates sister chromatid exchange and the fidelity of double-strand break repair, but not gene targeting, in plants transformed by Agrobacterium.

Expression of the bacterial RecA protein in plants stimulates homologous recombination in tobacco. Here we show that RecA plays a direct role in DNA strand exchange in vivo. The number of sister chromatid exchanges (SCEs) was increased 2.4-fold over wild type in transgenic tobacco plants expressing a nuclear-targeted RecA (nt-RecA) protein and could not be increased further by DNA damage, which caused a doubling of the baseline SCE frequency in wild-type plants. Although gene targeting requires homologous recombination, the number of targeted gene replacements was not increased markedly by the presence of nt-RecA by using Agrobacterium-mediated transformation. However, the number of double-strand breaks that were repaired at both sides by homologous recombination was increased 3.3-fold. Stimulation of SCE and fidelity of double-strand break repair by nt-RecA, but not by gene targeting, suggests that the stimulatory activity of RecA is linked to active DNA synthesis. Therefore, nascent replication-associated single strands may be a prerequisite for RecA action in plant cells.

RecA stimulates sister chromatid exchange and the fidelity of double-strand break repair, but not gene targeting, in plants transformed by Agrobacterium

Expression of the bacterial RecA protein in plants stimulates homologous recombination in tobacco. Here we show that RecA plays a direct role in DNA strand exchange in vivo. The number of sister chromatid exchanges (SCEs) was increased 2.4-fold over wild type in transgenic tobacco plants expressing a nuclear-targeted RecA (nt-RecA) protein and could not be increased further by DNA damage, which caused a doubling of the baseline SCE frequency in wild-type plants. Although gene targeting requires homologous recombination, the number of targeted gene replacements was not increased markedly by the presence of nt-RecA by using Agrobacterium-mediated transformation. However, the number of double-strand breaks that were repaired at both sides by homologous recombination was increased 3.3-fold. Stimulation of SCE and fidelity of double-strand break repair by nt-RecA, but not by gene targeting, suggests that the stimulatory activity of RecA is linked to active DNA synthesis. Therefore, nascent replication-associated single strands may be a prerequisite for RecA action in plant cells.

Solution structure of DinI provides insight into its mode of RecA inactivation

The Escherichia coli RecA protein triggers both DNA repair and mutagenesis in a process known as the SOS response. The 81-residue E. coli protein DinI inhibits activity of RecA in vivo. The solution structure of DinI has been determined by multidimensional triple resonance NMR spectroscopy, using restraints derived from two sets of residual dipolar couplings, obtained in bicelle and phage media, supplemented with J couplings and a moderate number of NOE restraints. DinI has an alpha/beta fold comprised of a three-stranded beta-sheet and two alpha-helices. The beta-sheet topology is unusual: the central strand is flanked by a parallel and an antiparallel strand and the sheet is remarkably flat. The structure of DinI shows that six negatively charged Glu and Asp residues on DinI's kinked C-terminal alpha-helix form an extended, negatively charged ridge. We propose that this ridge mimics the electrostatic character of the DNA phospodiester backbone, thereby enabling DinI to compete with single-stranded DNA for RecA binding. Biochemical data confirm that DinI is able to displace ssDNA from RecA.

Intersubunit proximity of residues in the RecA protein as shown by engineered disulfide cross-links.

Mutational studies of regions that make up the oligomeric interface within the RecA protein filament structure have shown that F217 is an important determinant of RecA function and oligomer stability. All substitutions, other than Tyr and Cys, completely inhibit RecA activities and exhibit a substantial decrease in protein filament stability [Skiba, M. C., and Knight, K. L. (1994) J. Biol. Chem. 269, 3823-3828; Logan, K. M., et al. (1997) J. Mol. Biol. 266, 306-316]. Although the RecA crystal structure exhibits no obvious constraints that explain this mutational stringency, the structure does reveal a hydrophobic pocket in the neighboring monomer that may accommodate the F217 side chain. Together with the F217C mutation, we have introduced a series of Cys substitutions within the interacting surface on the neighboring monomer and have tested for disulfide formation under various conditions, e.g., with or without ATP and ssDNA. We show that the location of F217 in the crystal structure is in general agreement with its position in the catalytically active RecA-ATP-DNA complex. Functional studies with the mutant proteins support the idea that ATP-induced movement of the wild-type F217 side chain toward this hydrophobic pocket is important in mediating allosteric changes in the RecA protein structure.

Saturation mutagenesis of the E. coli RecA loop L2 homologous DNA pairing region reveals residues essential for recombination and recombinational repair

The disordered mobile loop L2 of the Escherichia coli RecA protein is known to play a central role in DNA binding and pairing. To investigate the local chemical environment in relation to function we performed saturation mutagenesis of the loop L2 region (amino acid positions 193-212) using a site-directed mutagenesis procedure, and determined the recombinational proficiency of the 380 mutants using genetic assays for homologous recombination and recombinational repair. Residues Asn193, Gln194, Arg196, Glu207, Thr209, Gly211, and Gly212 were identified as stringently required for recombinational events in bacterial cells. In addition, our findings suggest the involvement of loop L2 in the ATPase activity of RecA, and a role for residues Gln194, Arg196, Lys198 and Thr209 in the DNA-dependent hydrolysis of ATP. Finally, since 20 residue peptides that comprise this region can pair homologous DNAs by forming filamentous β-structures, we propose how the information from the mutant analysis might facilitate the use of a simplified amino acid alphabet to design β-structure forming L2 peptides with improved RecA-like activities.

On the in vivo function of the RecA ATPase

The Escherichia coli RecA protein is the prototype of the RecA/RAD51/DMC1 family of strand transferases acting in genetic recombination. The E96D mutant was previously isolated in a screen for toxicrecA mutants and was found to constitutively derepress the SOS genes and inhibit chromosome segregation in E. coli. Here, we have found that the E96D mutation lowers the RecA kcatvalue for ATP hydrolysis 100-fold. Use of this mutant reveals that the ATPase and branch migration activities of RecA are not necessarily required for catalyzing in vivo recombinational pairing and LexA cleavage. In addition to its effect on ATP hydrolysis, the mutation causes ATP to more strongly promote the transition to the biologically active, extended conformation of the RecA enzyme. The enhanced ATP binding is apparently the cause for a broader nucleic acid ligand specificity. The use of RNA and double-stranded DNA as cofactors for LexA cleavage could give rise to the inappropriate, constitutive derepression of the SOS genes. This underscores the need for the ATP affinity to be optimized so that RecA becomes selectively activated only during DNA repair and recombination through binding single-stranded DNA.

Crystallization of Escherichia coli RuvA complexed with a synthetic Holliday junction.

During homologous recombination in Escherichia coli the RuvA, B and C proteins interact specifically with the Holliday junction formed by the action of RecA to promote the strand-exchange reaction. RuvA, a homotetrameric protein of molecular weight 88 kDa, has been overexpressed in E. coli, purified and co-crystallized with a synthetic Holliday junction substrate made from four 18-base deoxyoligonucleotides. Crystals were grown using the hanging-drop vapour-diffusion method with sodium acetate as the precipitant. The crystals diffract to a resolution of 6 A and belong to the monoclinic system, space group C2, with cell parameters a = 148, b = 148, c = 106 A and beta = 123 degrees. The X-ray analysis of these crystals should reveal the structure of the Holliday junction and its mode of binding to RuvA, providing new insights into the molecular mechanism of genetic recombination.

Characterization of the oligomeric states of RecA protein: monomeric RecA protein can form a nucleoprotein filament.

Self-assembly of RecA protein in solution and on single-stranded DNA exerts a significant effect on the catalytic activities of this protein. To manipulate the self-association reaction, we examined the effects of various salts on the self-association of RecA from Thermus thermophilus (ttRecA) by circular dichroism spectroscopy and gel-filtration analysis. We showed that the self-association of ttRecA strongly depends on the kind and concentration of the salt, as well as on the protein concentration. Chaotropic ions were especially useful for obtaining RecA in its hexameric and monomeric states. On the basis of these observations, we were able to regulate the oligomeric states of ttRecA and we then examined the activity of RecA in various oligomeric states. Monomeric ttRecA bound to ssDNA and formed a nucleoprotein filament, which showed ssDNA-dependent ATPase activity. These results suggest that the monomeric form of RecA is an intermediate in filament formation on ssDNA.