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.     

Biochemical events essential to the recombination activity of Escherichia coli RecA protein. II. Co-dominant effects of RecA142 protein on wild-type RecA protein function

In the accompanying paper, RecA142 protein was found to be completely defective in DNA heteroduplex formation. Here, we show that RecA142 protein not only is defective in this activity but also is inhibitory for certain activities of wild-type RecA protein. Under appropriate conditions, RecA142 protein substantially inhibits the DNA strand exchange reaction catalyzed by wild-type RecA protein; at equimolar concentrations of each protein, formation of full-length gapped duplex DNA product molecules is less than 7% of the amount produced by wild-type protein alone. Inhibition by RecA142 protein is also evident in S1 nuclease assays of DNA heteroduplex formation, although the extent of inhibition is less than is observed for the complete DNA strand exchange process; at equimolar concentrations of wild-type and mutant proteins, the extent of DNA heteroduplex formation is 36% of the wild-type protein level. This difference implies that RecA142 protein prevents, at minimum, the branch migration normally observed during DNA strand exchange. RecA142 protein does not inhibit either the single-strand (ss) DNA-dependent ATPase activity or the coaggregation activities of wild-type RecA protein. This suggests that these reactions are not responsible for the inhibition of wild-type protein DNA strand exchange activity by RecA142 protein. However, under conditions where RecA142 protein inhibits DNA strand exchange activity, RecA142 protein renders the M13 ssDNA-dependent ATPase activity of wild-type protein sensitive to inhibition by single-strand DNA-binding protein, and it inhibits the double-strand DNA-dependent ATPase activity of wild-type RecA protein. These results imply that these two activities are important components of the overall DNA strand exchange process. These experiments also demonstrate the applicability of using defective mutant RecA proteins as specific codominant inhibitors of wild-type protein activities in vitro and should be of general utility for mechanistic analysis of RecA protein function both in vitro and in vivo.     

Inhibition of RecA protein function by the RdgC protein from Escherichia coli

The Escherichia coli RdgC protein is a potential negative regulator of RecA function. RdgC inhibits RecA protein-promoted DNA strand exchange, ATPase activity, and RecA-dependent LexA cleavage. The primary mechanism of RdgC inhibition appears to involve a simple competition for DNA binding sites, especially on duplex DNA. The capacity of RecA to compete with RdgC is improved by the DinI protein. RdgC protein can inhibit DNA strand exchange catalyzed by RecA nucleoprotein filaments formed on single-stranded DNA by binding to the homologous duplex DNA and thereby blocking access to that DNA by the RecA nucleoprotein filaments. RdgC protein binds to single-stranded and double-stranded DNA, and the protein can be visualized on DNA using electron microscopy. RdgC protein exists in solution as a mixture of oligomeric states in equilibrium, most likely as monomers, dimers, and tetramers. This concentration-dependent change of state appears to affect its mode of binding to DNA and its capacity to inhibit RecA. The various species differ in their capacity to inhibit RecA function.     

Effects of the Escherichia coli SSB protein on the binding of Escherichia coli RecA protein to single-stranded DNA. Demonstration of competitive binding and the lack of a specific protein-protein interaction

The effect of the Escherichia coli single-stranded DNA binding (SSB) protein on the stability of complexes of E. coli RecA protein with single-stranded DNA has been investigated through direct DNA binding experiments. The effect of each protein on the binding of the other to single-stranded DNA, and the effect of SSB protein on the transfer rate of RecA protein from one single-stranded DNA molecule to another, were studied. The binding of SSB protein and RecA protein to single-stranded phage M13 DNA is found to be competitive and, therefore, mutually exclusive. In the absence of a nucleotide cofactor, SSB protein binds more tightly to single-stranded DNA than does RecA protein, whereas in the presence of ATP-gamma-S, RecA protein binds more tightly than SSB protein. In the presence of ATP, an intermediate result is obtained that depends on the type of DNA used, the temperature, and the magnesium ion concentration. When complexes of RecA protein, SSB protein and single-stranded M13 DNA are formed under conditions of slight molar excess of single-stranded DNA, no effect of RecA protein on the equilibrium stability of the SSB protein-single-stranded DNA complex is observed. Under similar conditions, SSB protein has no observed effect on the stability of the RecA protein-etheno M13 DNA complex. Finally, measurements of the rate of RecA protein transfer from RecA protein-single-stranded DNA complexes to competing single-stranded DNA show that there is no kinetic stabilization of the RecA protein-etheno M13 DNA complex by SSB protein, but that a tenfold stabilization is observed when single-stranded M13 DNA is used to form the complex. However, this apparent stabilizing effect of SSB protein can be mimicked by pre-incubation of the RecA protein-single-stranded M13 DNA complex in low magnesium ion concentration, suggesting that this effect of SSB protein is indirect and is mediated through changes in the secondary structure of the DNA. Since no direct effect of SSB protein is observed on either the equilibrium or dissociation properties of the RecA protein-single-stranded DNA complex, it is concluded that the likely effect of SSB protein in the strand assimilation reaction is on a slow step in the association of RecA protein with single-stranded DNA. Direct evidence for this conclusion is presented in the accompanying paper.     

A RecA filament capping mechanism for RecX protein

The RecX protein is a potent inhibitor of RecA protein activities. RecX functions by specifically blocking the extension of RecA filaments. In vitro, this leads to a net disassembly of RecA protein from circular single-stranded DNA. Based on multiple observations, we propose that RecX has a RecA filament capping activity. This activity has predictable effects on the formation and disassembly of RecA filaments. In vivo, the RecX protein may limit the length of RecA filaments formed during recombinational DNA repair and other activities. RecX protein interacts directly with RecA protein, but appears to interact in a functionally significant manner only with RecA filaments bound to DNA.     

RecA Protein from the extremely radioresistant bacterium Deinococcus radiodurans: expression, purification, and characterization

The RecA protein of Deinococcus radiodurans (RecA(Dr)) is essential for the extreme radiation resistance of this organism. The RecA(Dr) protein has been cloned and expressed in Escherichia coli and purified from this host. In some respects, the RecA(Dr) protein and the E. coli RecA (RecA(Ec)) proteins are close functional homologues. RecA(Dr) forms filaments on single-stranded DNA (ssDNA) that are similar to those formed by the RecA(Ec). The RecA(Dr) protein hydrolyzes ATP and dATP and promotes DNA strand exchange reactions. DNA strand exchange is greatly facilitated by the E. coli SSB protein. As is the case with the E. coli RecA protein, the use of dATP as a cofactor permits more facile displacement of bound SSB protein from ssDNA. However, there are important differences as well. The RecA(Dr) protein promotes ATP- and dATP-dependent reactions with distinctly different pH profiles. Although dATP is hydrolyzed at approximately the same rate at pHs 7.5 and 8.1, dATP supports an efficient DNA strand exchange only at pH 8.1. At both pHs, ATP supports efficient DNA strand exchange through heterologous insertions but dATP does not. Thus, dATP enhances the binding of RecA(Dr) protein to ssDNA and the displacement of ssDNA binding protein, but the hydrolysis of dATP is poorly coupled to DNA strand exchange. The RecA(Dr) protein thus may offer new insights into the role of ATP hydrolysis in the DNA strand exchange reactions promoted by the bacterial RecA proteins. In addition, the RecA(Dr) protein binds much better to duplex DNA than the RecA(Ec) protein, binding preferentially to double-stranded DNA (dsDNA) even when ssDNA is present in the solutions. This may be of significance in the pathways for dsDNA break repair in Deinococcus.     

Head to head dimer model; an alternative model for the strand exchange reaction by RecA protein of Escherichia coli

The RecA protein of E coli promotes a strand exchange reaction in vitro which appears to be similar to homologous genetic recombination in vivo. A model for the mechanism of strand transfer reaction by RecA protein has been proposed by Howard-Flanders et al based on the assumption that the RecA monomer has two distinctive DNA binding sites both of which can bind to ssDNA as well as dsDNA. Here, I propose an alternative model based on the assumption that RecA monomer has a single domain for binding to a polynucleotide chain with a unique polarity. In addition, the model is based on a few mechanical assumptions that, in the presence of ATP, two RecA molecules form a head to head dimer as the basic binding unit to DNA, and that the binding of RecA protein to a polynucleotide chain induces a structural change of RecA protein that causes a higher state of affinity for another RecA molecule that is expressed as cooperativy. The model explains many of the biochemical capabilities of RecA protein including the polar polymerization of RecA protein on single stranded DNA and polar strand transfer of DNA by the protein as well as the formation of a joint DNA molecule in a paranemic configuration. The model also presents the energetics in the strand transfer reaction.     

C-terminal truncated Escherichia coli RecA protein RecA5327 has enhanced binding affinities to single- and double-stranded DNAs.

RecA5327 is a truncated RecA protein that is lacking 25 amino acid residues from the C-terminal end. The expression of RecA5327 protein in the cell resulted in the constitutive induction of SOS functions without damage to the DNA. Purified RecA5327 protein effectively promoted the LexA repressor cleavage reaction and ATP hydrolysis at a lower concentration of single-stranded DNA than that required for wild-type RecA protein. A DNA binding study showed that RecA5327 has about ten times higher affinity for single-stranded DNA than does the wild-type RecA protein. Moreover RecA5327 protein binds stably to double-stranded (ds) DNA in conditions where the wild-type RecA protein could not bind. The binding of RecA5327 protein to dsDNA was associated with the unwinding of dsDNA, suggesting that RecA5327 binds to dsDNA in the same manner as does the wild-type protein. The fact that RecA5327 does not bind stoichiometrically but forms short filaments on dsDNA suggests that it nucleates to dsDNA much more frequently than does the wild-type protein. The role of the 25 C-terminal residues, in the regulation of RecA binding to DNA, is discussed.     

Targeting of a functional Escherichia coli RecA protein to the nucleus of plant cells

We have characterised a RecA protein fused to the simian virus 40 large T nuclear-localisation signal. The fusion protein was targeted to the nucleus in transgenic tobacco plants with high efficiency. By contrast, authentic RecA was not enriched in the nuclei of plant cells expressing comparable amounts of protein. For detailed characterisation of the strand-exchange activity of the nuclear-targeted RecA protein, a nearly identical protein was expressed in Escherichia coli and purified to homogeneity. This protein was found to bind to single-stranded DNA with the same stoichiometry and to promote the exchange of homologous DNA strands with the same kinetics as authentic RecA. It was concluded that the amino-terminal modification did not alter any of the essential properties of RecA and that the fusion protein is a fully functional strand-exchange protein. However, the ATPase activity of this protein was 20 times greater than that of RecA in the absence of single-stranded DNA. As with RecA, this activity was further stimulated by the addition of single-stranded DNA. Since ATPase activity is correlated with the ability of RecA to assume its high affinity state for DNA, the nuclear-targeted RecA protein might be regarded as a constitutively stimulated RecA variant, fully functional in promoting homologous recombination.     

The direction of RecA protein assembly onto single strand DNA is the same as the direction of strand assimilation during strand exchange

The RecA protein of Escherichia coli optimally promotes DNA strand exchange reactions in the presence of the single strand DNA-binding protein of E. coli (SSB protein). Under these conditions, assembly of RecA protein onto single-stranded DNA (ssDNA) occurs in three steps. First, the ssDNA is rapidly covered by SSB protein. The binding of RecA protein is then initiated by nucleation of a short tract of RecA protein onto the ssDNA. Finally, cooperative polymerization of additional RecA protein accompanied by displacement of SSB protein results in a ssDNA-RecA protein filament (Griffith, J. D., Harris, L. D., and Register, J. C. (1984) Cold Spring Harbor Symp. Quant. Biol. 49, 553-559). We report here that RecA protein assembly onto circular ssDNA yields RecA protein-covered circles in which greater than 85% are completely covered by RecA protein with no remaining SSB protein-covered segments (as detected by electron microscopy). However, when linear ssDNA is used, 90% of the filaments contain a short segment at one end complexed with SSB protein. This suggests that RecA protein assembly is unidirectional. Visualization of the assembly of RecA protein onto either long ssDNA tails (containing either 5' or 3' termini) or ssDNA gaps generated in double strand DNA allowed us to determine that the RecA protein polymerizes in the 5' to 3' direction on ssDNA and preferentially nucleates at ssDNA-double strand DNA junctions containing 5' termini.     

Effects of Escherichia coli SSB protein on the single-stranded DNA-dependent ATPase activity of Escherichia coli RecA protein: Evidence that SSB protein facilitates the binding of RecA protein to regions of secondary structure within single-stranded DNA

The effect that Escherichia coli single-stranded DNA binding (SSB) protein has on the single-stranded DNA-dependent ATPase activity of RecA protein is shown to depend upon a number of variables such as order of addition, magnesium concentration, temperature and the type of single-stranded DNA substrate used. When SSB protein is added to the DNA solution prior to the addition of RecA protein, a significant inhibition of ATPase activity is observed. Also, when SSB protein is added after the formation of a RecA protein-single-stranded DNA complex using either etheno M13 DNA, poly(dA) or poly(dT), or using single-stranded phage M13 DNA at lower temperature (25 °C) and magnesium chloride concentrations of 1 mm or 4 mm, a time-dependent inhibition of activity is observed. These results are consistent with the conclusion that SSB protein displaces the RecA protein from these DNA substrates, as described in the accompanying paper. However, if SSB protein is added last to complexes of RecA protein and single-stranded M13 DNA at elevated temperature (37 °C) and magnesium chloride concentrations of 4 mm or 10 mm, or to poly(dA) and poly(dT) that was renatured in the presence of RecA protein, no inhibition of ATPase activity is observed; in fact, a marked stimulation is observed for single-stranded M13 DNA. A similar effect is observed if the bacteriophage T4-coded gene 32 protein is substituted for SSB protein. The apparent stoichiometry of DNA (nucleotides) to RecA protein at the optimal ATPase activity for etheno M13 DNA, poly(dA) and poly(dT) is 6(±1) nucleotides per RecA protein monomer at 4 mm-MgCl₂ and 37 °C. Under the same conditions, the apparent stoichiometry obtained using single-stranded M13 DNA is 12 nucleotides per RecA protein monomer; however, the stoichiometry changes to 4.5 nucleotides per RecA protein monomer when SSB protein is added last. In addition, a stoichiometry of four nucleotides per RecA protein can be obtained with single-stranded M13 DNA in the absence of SSB protein if the reactions are carried out in 1 mm-MgCl₂. These data are consistent with the interpretation that secondary structure within the natural DNA substrate limits the accessibility of RecA protein to these regions. The role of SSB protein is to eliminate this secondary structure and allow RecA protein to bind to these previously inaccessible regions of the DNA. In addition, our results have disclosed an additional property of the RecA protein-single-stranded DNA complex: namely, in the presence of complementary base-pairing and at elevated temperatures and magnesium concentrations, a unique RecA protein-DNA complex forms that is resistant to inhibition by SSB protein.     

Analysis of double stranded DNA-dependent activities of Deinococcus radiodurans RecA protein

In this study, the double-stranded DNA-dependent activities of Deinococcus radiodurans RecA protein (Dr RecA) were characterized. The interactions of the Dr RecA protein with double-stranded DNA were determined, especially dsDNA-dependent ATP hydrolysis by the Dr RecA protein and the DNA strand exchange reaction, in which multiple branch points exist on a single RecA protein-DNA complex. A nucleotide cofactor (ATP or dATP ) was required for the Dr RecA protein binding to duplex DNA. In the presence of dATP, the nucleation step in the binding process occurred more rapidly than in the presence of ATP. Salts inhibited the binding of the Dr RecA protein to double-stranded DNA. Double-stranded DNA-dependent ATPase activities showed a different sensitivity to anion species. Glutamate had only a minimal effect on the double-stranded DNA-dependent ATPase activities, up to a concentration of 0.7 M. In the competition experiment for Dr RecA protein binding, the Dr RecA protein manifested a higher affinity to double-stranded DNA than was observed for single-stranded DNA.     

Induction of SOS functions: Regulation of proteolytic activity of E. coil RecA protein by interaction with DNA and nucleoside triphosphate

Damage to cellular DNA or interruption of chromosomal DNA synthesis leads to induction of the SOS functions in E. coli. The immediate agent of induction is the RecA protein, which proteolytically cleaves and inactivates repressors, leading to induction of genes they control. RecA protein modified by tif mutations allows expression of SOS functions in the absence of inducing treatments. We show here that tif-mutant RecA protein is more efficient than wild-type RecA protein in interacting with DNA and nucleoside triphosphate. This result suggests that formation of a complex with DNA and nucleoside triphosphate is the critical event that activates RecA protein to destroy repressors after SOS-inducing treatments, and that damage to cellular DNA promotes this reaction by providing single-stranded DNA or an active nucleoside triphosphate or both. Since dATP is the most effective nucleoside triphosphate in promoting repressor cleavage, we suggest that it is the natural cofactor of RecA protein in vivo.     

RNA-DNA hybridization promoted by E. coli RecA protein.

RecA protein of E. coli plays a central regulatory role that is induced by damage to DNA and results in the inactivation of LexA repressor. In vitro, RecA protein binds preferentially to single-stranded DNA to form a nucleoprotein filament that can recognize homology in naked duplex DNA and promote extensive strand exchange. Although RecA protein shows little tendency at neutral pH to bind to RNA, we found that it nonetheless catalyzed at 37 degrees C the hybridization of complementary RNA and single-stranded DNA sequences. Hybrids made by RecA protein at 37 degrees C appeared indistinguishable from ones prepared by thermal annealing. RNA-DNA hybridization by RecA protein at neutral pH required, as does RecA-promoted homologous pairing, optimal conditions for the formation of RecA nucleoprotein filaments. The cosedimentation of RNA with those filaments further paralleled observations made on the formation of networks of nucleoprotein filaments with double-stranded DNA, an instrumental intermediate in homologous pairing in vitro. These similarities with the pairing reaction support the view that RecA protein acts specifically in the hybridization reaction.     

Interaction of RecA protein with pBR322 DNA modified by N-hydroxy-2-acetylaminofluorene and 4-hydroxyaminoquinoline 1-oxide.

Interaction of RecA protein of Escherichia coli with pBR322 DNA modified by N-hydroxy-2-acetylaminofluorene (N-OH-AAF) and 4-hydroxyaminoquinoline 1-oxide (4HAQO) was investigated. RecA protein bound more efficiently to modified DNA than to unmodified DNA as judged by filter-binding and gel electrophoresis assay. The binding of RecA protein with modified DNA resulted in the stimulation of ATPase activity and the activation for RecA protein to stimulate the repressor cleavage. These abilities of RecA protein were increased proportionally to the number of adducts in the plasmid DNA (0-5 adducts). Apurinic and alkylated DNA did not activate RecA protein. We suggest that modification of DNA by N-OH-AAF and 4HAQO provides binding sites for RecA protein and may act as an activation signal for SOS response.     

Binding of double-stranded DNA by Escherichia coli RecA protein monitored by a fluorescent dye displacement assay.

We have developed a new assay to characterize the double-stranded DNA (dsDNA) binding properties of RecA protein. This assay is based on measurement of changes in the fluorescence of a 4',6-diamidino-2-phenylindole (DAPI)-dsDNA complex upon RecA protein binding. The binding of RecA protein to a complex of DAPI and dsDNA results in displacement of the bound DAPI, producing a decrease in the observed fluorescence. DAPI displacement is dependent on both RecA protein and ATP; dATP and, to a lesser extent, UTP and dCTP also support the DAPI displacement reaction, but dGTP, GTP, dITP and TTP do not. Binding stoichiometry for the RecA protein-dsDNA complex measured by DAPI displacement is 3 bp per RecA protein monomer in the presence of ATP. These results, taken together with data for mutant RecA proteins, suggest that this DAPI displacement assay monitors formation of the high affinity DNA binding state of RecA protein. Since this state of RecA protein defines the form of the nucleoprotein filament that is active in DNA strand exchange, these findings raise the possibility that the RecA protein-dsDNA filament may possess a homologous pairing capacity.     

RecA protein and SOS. Correlation of mutagenesis phenotype with binding of mutant RecA proteins to duplex DNA and LexA cleavage

The RecA protein of Escherichia coli is required for SOS-induced mutagenesis in addition to its recombinational and regulatory roles. We have suggested that RecA might participate directly in targeted mutagenesis by binding preferentially to the site of the DNA damage (e.g. pyrimidine dimer) because of its partially unwound nature; DNA polymerase III will then encounter RecA-coated DNA at the lesion and might replicate across the damaged site more often but with reduced fidelity. In support of this proposal, we have found that the phenotype of wild-type and mutant RecA for mutagenesis correlates with capacity to bind to double-stranded DNA. Wild-type RecA binds more efficiently to ultraviolet (u.v.)-irradiated, duplex DNA than to non-irradiated DNA. The RecA441 (Tif) protein that is constitutive for mutagenesis binds extremely well to double-stranded DNA with no lesions, whereas the RecA430 protein that is defective in mutagenesis binds poorly even to u.v.-irradiated DNA. The RecA phenotype also correlates with capacity to use duplex DNA as a cofactor for cleavage of the LexA repressor protein for SOS-controlled operons. Wild-type RecA provides efficient cleavage of LexA only with u.v.-irradiated duplex DNA; RecA441 cleaves well with non-irradiated DNA; RecA430 gives very poor cleavage even with u.v.-irradiated DNA. We conclude that the interaction of RecA with damaged double-stranded DNA is likely to be a critical component of SOS mutagenesis and to define a pathway for the LexA cleavage reaction as well.     

Targeting of a functional Escherichia coli RecA protein to the nucleus of plant cells

 We have characterised a RecA protein fused to the simian virus 40 large T nuclear-localisation signal. The fusion protein was targeted to the nucleus in transgenic tobacco plants with high efficiency. By contrast, authentic RecA was not enriched in the nuclei of plant cells expressing comparable amounts of protein. For detailed characterisation of the strand-exchange activity of the nuclear-targeted RecA protein, a nearly identical protein was expressed in Escherichia coli and purified to homogeneity. This protein was found to bind to single-stranded DNA with the same stoichiometry and to promote the exchange of homologous DNA strands with the same kinetics as authentic RecA. It was concluded that the amino-terminal modification did not alter any of the essential properties of RecA and that the fusion protein is a fully functional strand-exchange protein. However, the ATPase activity of this protein was 20 times greater than that of RecA in the absence of single-stranded DNA. As with RecA, this activity was further stimulated by the addition of single-stranded DNA. Since ATPase activity is correlated with the ability of RecA to assume its high affinity state for DNA, the nuclear-targeted RecA protein might be regarded as a constitutively stimulated RecA variant, fully functional in promoting homologous recombination. Received: 29 July 1996 / Accepted: 24 September 1996     

A DNA pairing-enhanced conformation of bacterial RecA proteins

The RecA proteins of Escherichia coli (Ec) and Deinococcus radiodurans (Dr) both promote a DNA strand exchange reaction involving two duplex DNAs. The four-strand exchange reaction promoted by the DrRecA protein is similar to that promoted by EcRecA, except that key parts of the reaction are inhibited by Ec single-stranded DNA-binding protein (SSB). In the absence of SSB, the initiation of strand exchange is greatly enhanced by dsDNA-ssDNA junctions at the ends of DNA gaps. This same trend is seen with the EcRecA protein. The results lead to an expansion of published hypotheses for the pathway for RecA-mediated DNA pairing, in which the slow first order step (observed in several studies) involves a structural transition to a state we designate P. The P state is identical to the state found when RecA is bound to double-stranded (ds) DNA. The structural state present when the RecA protein is bound to single-stranded (ss) DNA is designated A. The DNA pairing model in turn facilitates an articulation of three additional conclusions arising from the present work. 1) When a segment of a RecA filament bound to ssDNA is forced into the P state (as RecA bound to the ssDNA immediately adjacent to dsDNA-ssDNA junction), the segment becomes "pairing enhanced." 2) The unusual DNA pairing properties of the D. radiodurans RecA protein can be explained by postulating this protein has a more stringent requirement to initiate DNA strand exchange from the P state. 3) RecA filaments bound to dsDNA (P state) have directly observable structural changes relative to RecA filaments bound to ssDNA (A state), involving the C-terminal domain.     

Removal of the RecA C-terminus results in a conformational change in the RecA-DNA filament

The Escherichia coli RecA protein catalyzes homologous recombination of DNA molecules, and the active form of the protein is a helical polymer that it forms around DNA. Previous image analysis of electron micrographs has revealed the RecA protein to be organized into two domains or lobes within the RecA-DNA filament. We have now been able to show that a small modification of the RecA protein by proteolysis results in a significant shift in the internal mass in the RecA filament. We have cleaved approximately 18 residues from the C-terminus of the RecA protein, producing a roughly 36K MW RecA core protein that binds DNA and polymerizes normally. A three-dimensional reconstruction of this complex has been computed, and has been compared with a previous reconstruction of the intact protein. The main difference is consistent with a 15 A outward movement of the lobe that was at an inner radius in the wild-type protein. These observations yield additional evidence about the conformational flexibility of the RecA filament, and will aid in understanding the structural mechanics and dynamics of the RecA filament.