Binding of SSB and RecA protein to DNA-containing stem loop structures: SSB ensures the polarity of RecA polymerization on single-stranded DNA

Single-stranded DNA-binding proteins play an important role in homologous pairing and strand exchange promoted by the class of RecA-like proteins. It is presumed that SSB facilitates binding of RecA on to ssDNA by melting secondary structure, but direct physical evidence for the disruption of secondary structure by either SSB or RecA is still lacking. Using a series of oligonucleotides with increasing amounts of secondary structure, we show that stem loop structures impede contiguous binding of RecA and affect the rate of ATP hydrolysis. The electrophoretic mobility shift of a ternary complex of SSB-DNA-RecA and a binary complex of SSB-DNA are similar; however, the mechanism remains obscure. Binding of RecA on to stem loop is rapid in the presence of SSB or ATPgammaS and renders the complex resistant to cleavage by HaeIII, to higher amounts of competitor DNA or low temperature. The elongation of RecA filament in a 5' to 3' direction is halted at the proximal end of the stem. Consequently, RecA nucleates at the loop and cooperative binding propagates the RecA filament down the stem causing its disruption. The pattern of modification of thymine residues in the loop region indicates that RecA monomer is the minimum binding unit. Together, these results suggest that SSB plays a novel role in ensuring the directionality of RecA polymerization across stem loop in ssDNA. These observations have fundamental implications on the role of SSB in multiple aspects of cellular DNA metabolism.     

RecA protein-promoted homologous pairing and strand exchange between intact and partially single-stranded duplex DNA.

In the pairing reaction between circular gapped and fully duplex DNA, RecA protein first polymerizes on the gapped DNA to form a nucleoprotein filament. Conditions that removed the formation of secondary structure in the gapped DNA, such as addition of Escherichia coli single-stranded DNA binding protein or preincubation in 1 mM-MgCl2, optimized the binding of RecA protein and increased the formation of joint molecules. The gapped duplex formed stable joints with fully duplex DNA that had a 5' or 3' terminus complementary to the single-stranded region of the gapped molecule. However, the joints formed had distinct properties and structures depending on whether the complementary terminus was at the 5' or 3' end. Pairing between gapped DNA and fully duplex linear DNA with a 3' complementary terminus resulted in strand displacement, symmetric strand exchange and formation of complete strand exchange products. By contrast, pairing between gapped and fully duplex DNA with a 5' complementary terminus produced a joint that was restricted to the gapped region; there was no strand displacement or symmetric strand exchange. The joint formed in the latter reaction was likely a three-stranded intermediate rather than a heteroduplex with the classical Watson-Crick structure. We conclude that, as in the three-strand reaction, the process of strand exchange in the four-strand reaction is polar and progresses in a 5' to 3' direction with respect to the initiating strand. The present study provides further evidence that in both three-strand and four-strand systems the pairing and strand exchange reactions share a common mechanism.     

RecA protein filaments disassemble in the 5' to 3' direction on single-stranded DNA

RecA protein forms filaments on both single- and double-stranded DNA. Several studies confirm that filament extension occurs in the 5' to 3' direction on single-stranded DNA. These filaments also disassemble in an end-dependent fashion, and several indirect observations suggest that the disassembly occurs on the end opposite to that at which assembly occurs. By labeling the 5' end of single-stranded DNA with a segment of duplex DNA, we demonstrate unambiguously that RecA filaments disassemble uniquely in the 5' to 3' direction.     

The C terminus of the Escherichia coli RecA protein modulates the DNA binding competition with single-stranded DNA-binding protein

The nucleation step of Escherichia coli RecA filament formation on single-stranded DNA (ssDNA) is strongly inhibited by prebound E. coli ssDNA-binding protein (SSB). The capacity of RecA protein to displace SSB is dramatically enhanced in RecA proteins with C-terminal deletions. The displacement of SSB by RecA protein is progressively improved when 6, 13, and 17 C-terminal amino acids are removed from the RecA protein relative to the full-length protein. The C-terminal deletion mutants also more readily displace yeast replication protein A than does the full-length protein. Thus, the RecA protein has an inherent and robust capacity to displace SSB from ssDNA. However, the displacement function is suppressed by the RecA C terminus, providing another example of a RecA activity with C-terminal modulation. RecADeltaC17 also has an enhanced capacity relative to wild-type RecA protein to bind ssDNA containing secondary structure. Added Mg(2+) enhances the ability of wild-type RecA and the RecA C-terminal deletion mutants to compete with SSB and replication protein A. The overall binding of RecADeltaC17 mutant protein to linear ssDNA is increased further by the mutation E38K, previously shown to enhance SSB displacement from ssDNA. The double mutant RecADeltaC17/E38K displaces SSB somewhat better than either individual mutant protein under some conditions and exhibits a higher steady-state level of binding to linear ssDNA under all conditions.     

Fibers of RecA protein and complexes of RecA protein and single-stranded phi X174 DNA as visualized by negative-stain electron microscopy

Monomers of purified RecA protein polymerize into helical fibers whose pitch is 7.2 nm to 7.5 nm and whose diameter is 11 nm. Either short (approximately 0.2 micron), single fibers, or bundles of aligned, longer fibers, can be formed preferentially, by varying the Mg2+ concentration. When RecA protein is bound to circular, single-stranded phi X174 DNA it forms helical fibers of different classes of contour lengths, ranging from 0.98 micron, depending upon the conditions of assembly. Two different helical pitches are found, one of 9.3 nm when the incubation buffer contains, besides the obligatory Mg2+, either ATP gamma S or ATP accompanied by single-strand binding protein, and one of 5.5 nm when the latter additives are omitted. Preformed fibers of the compact type can be converted to open ones of 9.3 nm pitch upon addition of ATP gamma S, even after the removal of unbound RecA. All signs of helicity are obliterated upon glutaraldehyde cross-linking except in those fibers whose assembly has been mediated by ATP gamma S. RecA protein and single-strand binding protein are competitively bound to single-stranded DNA. Composite complexes, however, are not encountered unless ATP gamma S is present. Otherwise, segments of DNA that are coated by one or the other protein are seen as separate regions. When the assembly of complexes of single-stranded DNA and RecA is mediated by single-strand binding protein and ATP, the axial separation between successive bases is 0 X 42 nm, somewhat greater than the axial distance between bases in one strand of duplex DNA in the B form. It is proposed that the bases of the single-stranded DNA in the complex are located near its inner surface, and that base-pairing with double-stranded DNA takes place following invasion of the central cavity of the complex.     

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.     

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.     

Characterization of thermostable RecA protein and analysis of its interaction with single-stranded DNA.

Thermostable RecA protein (ttRecA) from Thermus thermophilus HB8 showed strand exchange activity at 65 degrees C but not at 37 degrees C, although nucleoprotein complex was observed at both temperatures. ttRecA showed single-stranded DNA (ssDNA)-dependent ATPase activity, and its activity was maximal at 65 degrees C. The kinetic parameters, K(m) and kcat, for adenosine triphosphate (ATP) hydrolysis with poly(dT) were 1.4 mM and 0.60 s-1 at 65 degrees C, and 0.34 mM and 0.28 s-1 at 37 degrees C, respectively. Substrate cooperativity was observed at both temperatures, and the Hill coefficient was about 2. At 65 degrees C, all tested ssDNAs were able to stimulate the ATPase activity. The order of ATPase stimulation was: poly(dC) > poly(dT) > M13 ssDNA > poly(dA). Double-stranded DNAs (dsDNA), poly(dT).poly(dA) and M13 dsDNA, were unable to activate the enzyme at 65 degrees C. At 37 degrees C, however, not only dsDNAs but also poly(dA) and M13 ssDNA showed poor stimulating ability. At 25 degrees C, poly(dA) and M13 ssDNA gave circular dichroism (CD) peaks at around 192 nm, which reflect a particular structure of DNA. The conformation was changed by an upshift of temperature or binding to Escherichia coli RecA protein (ecRecA), but not to ttRecA. The dissociation constant between ecRecA and poly(dA) was estimated to be 44 microM at 25 degrees C by the change in the CD. These observations suggest that the capability to modify the conformation of ssDNA may be different between ttRecA and ecRecA. The specific structure of ssDNA was altered by heat or binding of ecRecA. After this alteration, ttRecA and ecRecA can express their activities at each physiological temperature.     

Assembly of presynaptic filaments. Factors affecting the assembly of RecA protein onto single-stranded DNA

We have previously shown that the assembly of RecA protein onto single-stranded DNA (ssDNA) facilitated by SSB protein occurs in three steps: (1) rapid binding of SSB protein to the ssDNA; (2) nucleation of RecA protein onto this template; and (3) co-operative polymerization of additional RecA protein to yield presynaptic filaments. Here, electron microscopy has been used to further explore the parameters of this assembly process. The optimal extent of presynaptic filament formation required at least one RecA protein monomer per three nucleotides, high concentrations of ATP (greater than 3 mM in the presence of 12 mM-Mg2+), and relatively low concentrations of SSB protein (1 monomer per 18 nucleotides). Assembly was depressed threefold when SSB protein was added to one monomer per nine nucleotides. These effects appeared to be exerted at the nucleation step. Following nucleation, RecA protein assembled onto ssDNA at net rates that varied from 250 to 900 RecA protein monomers per minute, with the rate inversely related to the concentration of SSB protein. Combined sucrose sedimentation and electron microscope analysis established that SSB protein was displaced from the ssDNA during RecA protein assembly.     

Equilibrium binding of single-stranded DNA to the secondary DNA binding site of the bacterial recombinase RecA

The bacterial recombinase RecA forms a nucleoprotein filament in vitro with single-stranded DNA (ssDNA) at its primary DNA binding site, site I. This filament has a second site, site II, which binds ssDNA and double-stranded DNA. We have investigated the binding of ssDNA to the RecA protein in the presence of adenosine 5'-O-(thiotriphosphate) cofactor using fluorescence anisotropy. The RecA protein carried out DNA strand exchange with a 5'-fluorescein-labeled 32-mer oligonucleotide. The anisotropy signal was shown to measure oligonucleotide binding to RecA, and the relationship between signal and binding density was determined. Binding of ssDNA to site I of RecA was stable at high NaCl concentrations. Binding to site II could be described by a simple two-state equilibrium, K = 4.5 +/- 1.5 x 10(5) m(-1) (37 degrees C, 150 mm NaCl, pH 7.4). The reaction was enthalpy-driven and entropy-opposed. It depended on salt concentration and was sensitive to the type of monovalent anion, suggesting that anion-dependent protein conformations contribute to ssDNA binding at site II.     

Force and ATP hydrolysis dependent regulation of RecA nucleoprotein filament by single-stranded DNA binding protein

In Escherichia coli, the filament of RecA formed on single-stranded DNA (ssDNA) is essential for recombinational DNA repair. Although ssDNA-binding protein (SSB) plays a complicated role in RecA reactions in vivo, much of our understanding of the mechanism is based on RecA binding directly to ssDNA. Here we investigate the role of SSB in the regulation of RecA polymerization on ssDNA, based on the differential force responses of a single 576-nucleotide-long ssDNA associated with RecA and SSB. We find that SSB outcompetes higher concentrations of RecA, resulting in inhibition of RecA nucleation. In addition, we find that pre-formed RecA filaments de-polymerize at low force in an ATP hydrolysis- and SSB-dependent manner. At higher forces, re-polymerization takes place, which displaces SSB from ssDNA. These findings provide a physical picture of the competition between RecA and SSB under tension on the scale of the entire nucleoprotein SSB array, which have broad biological implications particularly with regard to competitive molecular binding.     

DNA recognition of a 24-mer peptide derived from RecA protein

A novel 24-residue peptide (L2-G), Ile-Arg-Met-Lys-Ile-Gly-Val-Met-Phe-Gly-Asn-Pro-Glu-Thr-Thr-Thr-Gly-Gly-Asn-Ala-Leu-Lys-Phe-Tyr, derived from RecA can discriminate a single-stranded DNA (ssDNA) from a double-stranded DNA (dsDNA) and a new developed support with this peptide recognizes not dsDNA but ssDNA. The 24-mer peptide with L2 and helix G amino acids of Escherichia coli RecA protein showed the ssDNA binding property with more than 1000 times affinity difference for the dsDNA. However, truncated 15-mer peptide showed no ssDNA binding activity. In the ssDNA binding, L2-G changed its conformation with the perturbation of an alpha-helix structure. The ssDNA binding and the DNA discrimination property of this peptide were due to almost all L2 and helix G amino acids, respectively. This result is useful to design synthetic peptides as functional materials for DNA recognition.     

RecA protein inhibits in vitro replication of single-stranded DNA with DNA polymerase III holoenzyme of Escherichia coli

Purified RecA protein from Escherichia coli inhibited 5-10-fold the rate of in vitro replication of both unirradiated and UV-irradiated single-stranded DNA (ssDNA) with DNA polymerase III holoenzyme. Maximal inhibition occurred at a ratio of 1 molecule of RecA per 2-4 nucleotides of DNA, and it affected primarily the initiation of elongation on primed ssDNA. Adding single-strand DNA-binding protein (SSB) caused a relief of inhibition. Under conditions when there was enough SSB to cover the ssDNA completely, RecA protein had no effect on initiation, elongation or dissociation steps of replication. These observations together with data from in vivo studies suggest a role for RecA protein in the arrest of DNA replication observed in cells exposed to UV-radiation and a variety of chemical carcinogens.     

Cofactor-induced orientation of the DNA bases in single-stranded DNA complexed with RecA protein. A fluorescence anisotropy and time-decay study

The structure of the RecA-single-stranded DNA complex was investigated by studying the fluorescence emission of poly(deoxy-1,N6-ethenoadenylic acid (poly(d epsilon A)), a fluorescent derivative of poly(dA), under various viscosity conditions. The fluorescence intensity and average lifetime of poly(d epsilon A) are much smaller than those of nonpolymerized monoethenonucleotides (1,N6-ethenoadenosine 5'-triphosphate and 1,N6-ethenoadenine deoxyribose 5'-monophosphate) at low viscosity and reflect intramolecular base-base collisions in the polymer. They considerably increased upon RecA binding, both in the presence and absence of cofactor ATP or adenosine 5'-O-(3-thiotriphosphate). This increase, as well as the increase in fluorescence anisotropy upon RecA binding, was very similar to that which resulted from sucrose addition to free poly(d epsilon A). These observations point to a decrease in the mobility of DNA bases upon RecA binding. In the presence of cofactor, the fluorescence features became independent of viscosity. This strongly suggests the absence of base motion of significant amplitude on the time scale of the fluorescence lifetime (about 10 ns). In the absence of cofactor, however, these features remained sensitive to viscosity, implying residual local motions of the bases. Such cofactor-dependent rigid attachment of DNA bases to stiff phosphate backbone could facilitate the search for homology between two DNA molecules during recombination.     

The hRad51 and RecA proteins show significant differences in cooperative binding to single-stranded DNA.

The human Rad51 protein (hRad51), like its bacterial homologue RecA, catalyzes genetic recombination between homologous single and double-stranded DNA substrates. Using IAsys biosensor technology, we have examined the critical first step in this process, the binding of hRad51 and RecA to ssDNA. We show that hRad51 binds cooperatively and with high affinity to an oligonucleotide substrate in both the absence and presence of nucleotide cofactors. In fact, both ATP and ATPgammaS have a slight inhibitory effect on hRad51 binding affinity. We show that this results from a decrease in the intrinsic affinity of a given monomer for ssDNA, which is counterbalanced by an increase in the cooperative assembly of protein onto DNA. In contrast, we show that the dramatic NTP-induced increase in ssDNA binding affinity of RecA is accounted for by a significant increase in cooperative filament assembly and not by an increase in the intrinsic DNA binding affinity of monomeric RecA. These results demonstrate that although the hRad51 and RecA proteins display many structural and functional similarities, they show profound inherent mechanistic differences.     

Recognition of oxidized thymine base on the single-stranded DNA by replication protein A

Replication protein A (RAP) is a eukaryotic single-stranded DNA binding protein involved in DNA replication, repair, and recombination. Recent studies indicate that RPA preferentially binds the damaged sites rather than the undamaged sites. Therefore, RPA is thought to be a member ofrepair factories or a sensor of lesion on DNA. To obtain further information of behavior of RPA against the oxidized lesion, we studied the binding affinity of RPA for the single-stranded DNA containing 5-formyluracil, a major lesion of thymine base yielded by the oxidation, using several synthetic oligonucleotides. The affinity of RPA for oligonucleotides was determined by gel shift assay. Results suggest that the surrounding sequence of 5-formyluracil may affect the affinity for RPA, and that the 5-formyluracil on the purine stretch but not the pyrimidine stretch increases the affinity for RPA. Results of affinity labeling experiment of RPA with the oligonucleotides containing 5-formyluracil indicate that RPA1 subunit may directly recognize and bind to the 5-formyluracil on the single-stranded DNA.     

The uvsX protein of bacteriophage T4 arranges single-stranded and double-stranded DNA into similar helical nucleoprotein filaments

The bacteriophage T4 uvsX gene codes for a DNA-binding protein that is important for genetic recombination in T4-infected cells. This protein is a DNA-dependent ATPase that resembles the Escherichia coli recA protein in many of its properties. We have examined the binding of purified uvsX protein to single-stranded DNA (ssDNA) and to double-stranded DNA (dsDNA) using electron microscopy to visualize the complexes that are formed and double label analysis to measure their protein content. We find that the uvsX protein binds cooperatively to dsDNA, forming filaments 14 nm in diameter with an apparently helical axial repeat of 12 nm. Each repeat contains about 42 base pairs and 9-12 uvsX protein monomers. In solutions containing Mg2+, the uvsX protein also binds cooperatively to ssDNA. The filaments that result are 14 nm in diameter, show a 12-nm axial repeat, and they are nearly identical in appearance to the filaments that contain dsDNA. In the filaments formed along ssDNA, each axial repeat contains about 49 DNA bases and 9-12 uvsX monomers. Both the filaments formed on the ssDNA and dsDNA show a strong tendency to align side-by-side. T4 gene 32 protein also binds cooperatively to ssDNA and interacts both physically and functionally with uvsX protein. However, when gene 32 and uvsX proteins were added to ssDNA together, no interaction between the two proteins was detected.     

Interaction of the RecA protein of Escherichia coli with single-stranded oligodeoxyribonucleotides.

The RecA protein of Escherichia coli performs a number of ATP-dependent, in vitro reactions and is a DNA-dependent ATPase. Small oligodeoxyribonucleotides were used as DNA cofactors in a kinetic analysis of the ATPase reaction. Polymers of deoxythymidilic acid as well as oligonucleotides of mixed base composition stimulated the RecA ATPase activity in a length-dependent fashion. Both the initial rate and the extent of the reaction were affected by chain length. Full activity was seen with chain lengths > or = 30 nt. Partial activity was seen with chain lengths of 15-30 nt. The lower activity of shorter oligonucleotides was not simply due to a reduced affinity for DNA, since effects of chain length on KmATP and the Hill coefficient for ATP hydrolysis were also observed. The results also suggested that single-stranded DNA secondary structure frequently affects the ATPase activity of RecA protein with oligodeoxyribonucleotides.     

New mutations in and around the L2 disordered loop of the RecA protein modulate recombination and/or coprotease activity.

The RecA protein plays a key role in Escherichia coli recombination and DNA repair. We have created new recA mutants with mutations in the vicinity of the recA430 mutation (Gly-204----Ser) which is known to affect RecA coprotease activity. Mutants carrying recA659 or recA611, located 3 and 7 amino acids downstream of residue 204, respectively, lose all RecA activities, while the mutant carrying recA616, which is located at 12 amino acids from this residue, keeps the coprotease activity but is unable to promote recombination. Complementation experiments show that both mutations recA611 and recA659 are dominant over the wild-type or recA430 allele while recA616 seems to be recessive to recA+ and dominant over recA430. It is suggested that these mutations are located in RecA domains which direct conformational modifications.