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.     

General mechanism for RecA protein binding to duplex DNA

RecA protein binding to duplex DNA occurs by a multi-step process. The tau analysis, originally developed to examine the binding of RNA polymerase to promoter DNA, is adapted here to study two kinetically distinguishable reaction segments of RecA-double stranded (ds) DNA complex formation in greater detail. One, which is probably a rapid preequilibrium in which RecA protein binds weakly to native dsDNA, is found to have the following properties: (1) a sensitivity to pH, involving a net release of approximately one proton; (2) a sensitivity to salts; (3) little or no dependence on temperature; (4) little or no dependence on DNA length. The second reaction segment, the rate-limiting nucleation of nucleoprotein filament formation accompanied by partial DNA unwinding, is found to have the following properties: (1) a sensitivity to pH, involving a net uptake of approximately three protons; (2) a sensitivity to salts; (3) a relatively large dependence on temperature, with an Arrhenius activation energy of 39 kcal mol(-1); (4) a sensitivity to DNA topology; (5) a dependence on DNA length. These results contribute to a general mechanism for RecA protein binding to duplex DNA, which can provide a rationale for the apparent preferential binding to altered DNA structures such as pyrimidine dimers and Z-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.     

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.     

The RecOR proteins modulate RecA protein function at 5' ends of single-stranded DNA.

The Escherichia coli RecF, RecO and RecR pro teins have previously been implicated in bacterial recombinational DNA repair at DNA gaps. The RecOR-facilitated binding of RecA protein to single-stranded DNA (ssDNA) that is bound by single-stranded DNA-binding protein (SSB) is much faster if the ssDNA is linear, suggesting that a DNA end (rather than a gap) facilitates binding. In addition, the RecOR complex facilitates RecA protein-mediated D-loop formation at the 5' ends of linear ssDNAs. RecR protein remains associated with the RecA filament and its continued presence is required to prevent filament disassembly. RecF protein competes with RecO protein for RecR protein association and its addition destabilizes RecAOR filaments. An enhanced function of the RecO and RecR proteins can thus be seen in vitro at the 5' ends of linear ssDNA that is not as evident in DNA gaps. This function is countered by the RecF/RecO competition for association with the RecR protein.     

Difference between active and inactive nucleotide cofactors in the effect on the DNA binding and the helical structure of RecA filament dissociation of RecA--DNA complex by inactive nucleotides.

The RecA protein requires ATP or dATP for its coprotease and strand exchange activities. Other natural nucleotides, such as ADP, CTP, GTP, UTP and TTP, have little or no activation effect on RecA for these activities. We have investigated the activation mechanism, and the selectivity for ATP, by studying the effect of various nucleotides on the DNA binding and the helical structure of the RecA filament. The interaction with DNA was investigated via fluorescence measurements with a fluorescent DNA analog and fluorescein-labeled oligonucleotides, assisted by linear dichroism. Filament structure was investigated via small-angle neutron scattering. There is no simple correlation between filament elongation, DNA binding affinity of RecA, and DNA structure in the RecA complex. There may be multiple conformations of RecA. Both coprotease and strand exchange activities require formation of a rigid and well organized complex. The triphosphate nucleotides which do not activate RecA, destabilize the RecA-DNA complex, indicating that the chemical nature of the nucleotide nucleobase is very important for the stability of RecA-DNA complex. Higher stability of the RecA-DNA complex in the presence of adenosine 5'-O-3-thiotriphosphate or guanosine 5'-O-3-thiotriphosphate than ATP or GTP indicates that contact between the protein and the chemical group at the gamma position of the nucleotide also affects the stability of the RecA-DNA complex. This contact appears also important for the rigid organization of DNA because ADP strongly decreases the rigidity of the complex.     

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.     

RecA-mediated annealing of single-stranded DNA and its relation to the mechanism of homologous recombination

We demonstrate that RecA protein can mediate annealing of complementary DNA strands in vitro by at least two different mechanisms. The first annealing mechanism predominates under conditions where RecA protein causes coaggregation of single-stranded DNA (ssDNA) molecules and where RecA-free ssDNA stretches are present on both reaction partners. Under these conditions annealing can take place between locally concentrated protein-free complementary sequences. Other DNA aggregating agents like histone H1 or ethanol stimulate annealing by the same mechanism. The second mechanism of RecA-mediated annealing of complementary DNA strands is best manifested when preformed saturated RecA-ssDNA complexes interact with protein-free ssDNA. In this case, annealing can occur between the ssDNA strand resident in the complex and the ssDNA strand that interacts with the preformed RecA-ssDNA complex. Here, the action of RecA protein reflects its specific recombination promoting mechanism. This mechanism enables DNA molecules resident in the presynaptic RecA-DNA complexes to be exposed for hydrogen bond formation with DNA molecules contacting the presynaptic RecA-DNA filament.     

Regulation in repressor inactivation by RecA protein

Treatments that damage DNA or inhibit DNA synthesis in E. coli induce the expression of a set of functions called SOS functions that are involved in DNA repair, mutagenesis, arrest of cell division and prophage induction. Induction of SOS functions is triggered by inactivation of the LexA repressor or a phage repressor. Inactivation of these repressors results from their cleavage by the E. coli RecA protein in the presence of single-stranded DNA and a nucleoside triphosphate.We found that these cleavage reactions are controlled by two mechanisms in vitro: one is through the structural change of the RecA protein in the ternary complex, RecA-ssDNA-ATP-γ-S. The active ternary complex is formed by binding of ATP-γ-S to a complex of RecA protein and ssDNA. On the other hand, when the RecA protein binds to ATP-γ-S prior to its binding to ssDNA, the resulting complex has no or only very weak cleavage activity toward the repressor. This structural change is negatively controlled by its C-terminal part. The loss of the 25 amino acid residues from the C-terminal leads the RecA protein to stable binding to dsDNA as well as ssDNA, and the protein takes the activated form for the repressor cleavage constitutively. The other mechanism is through the structural change of the repressor. The cleavage reaction of a ∅80cI repressor is greatly stimulated by the presence of d(G-G), and d(G-G) stimulates the cleavage by binding to the C-terminal half of the ∅80cI repressor. Moreover, the C-terminal fragment of the cleaved products of the 80cI repressor was able to cleave a ∅80cI-λ chimeric repressor. These results strongly suggested that th active site of the repressor cleavage was located in the C-terminal domain of the repressor and that the C-terminal fragment produced by the cleavage could cleave the repressor.     

The stretched DNA geometry of recombination and repair nucleoprotein filaments

The RecA protein of Escherichia coli plays essential roles in homologous recombination and restarting stalled DNA replication forks. In vitro, the protein mediates DNA strand exchange between single-stranded (ssDNA) and homologous double-stranded DNA (dsDNA) molecules that serves as a model system for the in vivo processes. To date, no high-resolution structure of the key intermediate, comprised of three DNA strands simultaneously bound to a RecA filament (RecA x tsDNA complex), has been elucidated by classical methods. Here we review the systematic characterization of the helical geometries of the three DNA strands of the RecA x tsDNA complex using fluorescence resonance energy transfer (FRET) under physiologically relevant solution conditions. Measurements of the helical parameters for the RecA x tsDNA complex are consistent with the hypothesis that this complex is a late, poststrand-exchange intermediate with the outgoing strand shifted by about three base pairs with respect to its registry with the incoming and complementary strands. All three strands in the RecA x tsDNA complex adopt extended and unwound conformations similar to those of RecA-bound ssDNA and dsDNA.     

Specific RecA amino acid changes affect RecA–UmuD'C interaction

The UmuD'C mutagenesis complex accumulates slowly and parsimoniously after a 12 J m⁻² UV flash to attain after 45 min a low cell concentration between 15 and 60 complexes. Meanwhile, RecA monomers go up to 72 000 monomers. By contrast, when the UmuD'C complex is constitutively produced at a high concentration, it inhibits recombinational repair and then markedly reduces bacterial survival from DNA damage. We have isolated novel recA mutations that enable RecA to resist UmuD'C recombination inhibition. The mutations, named recA [UmuR], are located on the RecA three-dimensional structure at three sites: (i) the RecA monomer tail domain (four amino acid changes); (ii) the RecA monomer head domain (one amino acid change, which appears to interface with the amino acids in the tail domain); and (iii) in the core of a RecA monomer (one amino acid change). RecA [UmuR] proteins make recombination more efficient in the presence of UmuD'C while SOS mutagenesis is inhibited. The UmuR amino acid changes are located at a head-tail joint between RecA monomers and some are free to possibly interact with UmuD'C at the tip of a RecA polymer. These two RecA structures may constitute possible sites to which the UmuD'C complex might bind, hampering homologous recombination and favouring SOS mutagenesis.     

Two forms of RecA-single-stranded DNA-adenosine 5'-O-(3-thiotriphosphate) complexes with different activities for cleavage of phage phi 80 cI repressor

The kinetics of cleavage of the phage phi 80 cI repressor by Escherichia coli RecA protein were studied. The rate of cleavage in the presence of single-stranded DNA (ssDNA) and either adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S), ATP or dATP is very low in the first hour at 37 degrees C and then increases sharply as incubation continues. The initial rate of cleavage of the repressor is greatly increased by incubating the RecA protein with ssDNA prior to addition of ATP gamma S and the repressor. However, when ATP gamma S is present during preincubation of RecA protein with ssDNA, the stimulatory effect of preincubation is greatly reduced. This difference in the effect of preincubation in two different conditions can be explained by formation of RecA-ssDNA-ATP gamma S complexes with different activities for cleavage of the repressor. The active complex is formed by binding of ATP gamma S to a complex of RecA protein and ssDNA. However, when the RecA protein binds to ATP gamma S prior to its binding to ssDNA, the resulting complex has no or only very weak cleavage activity toward the repressor.     

A recombinational defect in the C-terminal domain of Escherichia coli RecA2278-5 protein is compensated by protein binding to ATP

RecA2278-5 is a mutant RecA protein (RecAmut) bearing two amino acid substitutions, Gly-278 to Thr and Val-275 to Phe, in the α-helix H of the C-terminal sub-domain of the protein. recA2278-5 mutant cells are unusual in that they are thermosensitive for recombination but almost normal for DNA repair of UV damage and the SOS response. Biochemical analysis of purified RecAmut protein revealed that its temperature sensitivity is suppressed by prior binding of this protein to its ligand. In fact, the preheating of RecAmut protein for several minutes at a restrictive temperature (42°C) in the absence of ATP resulted in inhibition at 42°C of many activities related to homologous recombination including ss- and dsDNA binding, high-affinity binding for ATP, ss- or dsDNA-dependent ATPase, RecA–RecA interaction, and strand transfer capability. The binary complex RecAmut::ATP under the same conditions showed a decrease in only two activities, i.e. dsDNA binding and high-affinity binding for ATP. Besides ATP, sodium acetate (1.5M) was shown to be another factor that can stabilize the RecAmut protein at 42°C, judging by restoration of its DNA-free ATPase activity. The similarity of influence of high salt (with its non-specific binding) and ATP (binding specifically) on the apparent protein folding stability suggests that the structural stability of the RecA C-terminal domain is one of the conditions for correct interaction between RecA protein and ATP in the RecA::ATP::ssDNA presynaptic complex formation. The decrease in affinity for ATP was suggested to be the factor that determined a particular recombinational (but not repair) thermosensitivity of the RecAmut protein. Finally, we show that the stability of C-terminal domain appeared to be necessary for the dsDNA-binding activity of the protein.     

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.     

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.     

Conserved conformation of RecA protein after executing the DNA strand-exchange reaction. A site-specific linear dichroism structure study

RecA protein and its eukaryotic homologue Rad51 protein catalyzes the DNA strand exchange, which is a key reaction of homologous recombination. At the initial step of the reaction, RecA proteins form a helical filament on a single-stranded DNA (ssDNA). Binding of double-stranded DNA (dsDNA) to the filament triggers the homology search; as homology is found, the exchange of strands occurs, and the displaced DNA is released. These are the principal steps of genetic recombination; however, despite many years of extensive study of RecA activities, the details of the mechanism are still obscure. A high-resolution structure of the active nucleoprotein filament could provide information to help understand this process. Using a linear dichroism polarized-light spectroscopy technique, in combination with protein engineering (the site-specific linear dichroism method), we have previously studied the arrangement of RecA in complex with ssDNA. In the present study, we have used this approach to search for structural variations of RecA at the atomic level as the DNA in the complex is changed from ssDNA to dsDNA. The structural data of the RecA-dsDNA filament are found to be very similar to the data previously obtained for the RecA-ssDNA complex, indicating that the overall orientation and also the internal structure of RecA in the active filament are not markedly altered when the bound DNA changes from single- to double-stranded. The implications of the structural similarities as well as the significance of some conformational variations observed for a few amino acid residues that may be involved in interactions with DNA are discussed.