The effects on strand exchange of 5' versus 3' ends of single-stranded DNA in RecA nucleoprotein filaments

Since the ends of DNA chains are thought to be important in homologous recombination, the way in which RecA protein and similar recombination enzymes process ends is important. We analyzed the effects of ends both on the formation of joints, and the progression of strand exchange. When the only homologous end was provided by a single strand, there was no significant difference between the formation of joints at a 5' end or a 3' end; but in agreement with the report of Konforti & Davis, Escherichia coli single-stranded DNA binding protein (SSB) selectively inhibited the activity of 5' ends. Complete strand exchange, assessed by study of linear single-stranded and double-stranded substrates, took place only in the 5' to 3' direction relative to DNA in the nucleoprotein filament. These observations pose a paradox: in the presence of SSB, of which there are about 800 tetramers per cell, the formation of homologous joints by RecA protein is favored at a 3' end, from which, however, authentic strand exchange appears not to occur. Since observations reported here and elsewhere show that joints have different properties when formed at a 5' versus a 3' end, we suggest that they may be processed differently in vivo.     

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.     

SSB protein limits RecOR binding onto single-stranded DNA

The RecO and RecR proteins form a complex that promotes the nucleation of RecA protein filaments onto SSB protein-coated single-stranded DNA (ssDNA). However, even when RecO and RecR proteins are provided at optimal concentrations, the loading of RecA protein is surprisingly slow, typically proceeding with a lag of 10 min or more. The rate-limiting step in RecOR-promoted RecA nucleation is the binding of RecOR protein to ssDNA, which is inhibited by SSB protein despite the documented interaction between RecO and SSB. Full activity of RecOR is seen only when RecOR is preincubated with ssDNA prior to the addition of SSB. The slow binding of RecOR to SSB-coated ssDNA involves the C terminus of SSB. When an SSB variant that lacks the C-terminal 8 amino acids is used, the capacity of RecOR to facilitate RecA loading onto the ssDNA is largely abolished. The results are used in an expanded model for RecOR action.     

Polynucleotide phosphorylase exonuclease and polymerase activities on single-stranded DNA ends are modulated by RecN, SsbA and RecA proteins

Bacillus subtilis pnpA gene product, polynucleotide phosphorylase (PNPase), is involved in double-strand break (DSB) repair via homologous recombination (HR) or non-homologous end-joining (NHEJ). RecN is among the first responders to localize at the DNA DSBs, with PNPase facilitating the formation of a discrete RecN focus per nucleoid. PNPase, which co-purifies with RecA and RecN, was able to degrade single-stranded (ss) DNA with a 3' → 5' polarity in the presence of Mn(2+) and low inorganic phosphate (Pi) concentration, or to extend a 3'-OH end in the presence dNDP · Mn(2+). Both PNPase activities were observed in evolutionarily distant bacteria (B. subtilis and Escherichia coli), suggesting conserved functions. The activity of PNPase was directed toward ssDNA degradation or polymerization by manipulating the Pi/dNDPs concentrations or the availability of RecA or RecN. In its dATP-bound form, RecN stimulates PNPase-mediated polymerization. ssDNA phosphorolysis catalyzed by PNPase is stimulated by RecA, but inhibited by SsbA. Our findings suggest that (i) the PNPase degradative and polymerizing activities might play a critical role in the transition from DSB sensing to end resection via HR and (ii) by blunting a 3'-tailed duplex DNA, in the absence of HR, B. subtilis PNPase might also contribute to repair via NHEJ.     

Activation of DNA-dependent protein kinase by single-stranded DNA ends

DNA-dependent protein kinase (DNA-PK) is involved in joining DNA double-strand breaks induced by ionizing radiation or V(D)J recombination. The kinase is activated by DNA ends and composed of a DNA binding subunit, Ku, and a catalytic subunit, DNA-PK(CS). To define the DNA structure required for kinase activation, we synthesized a series of DNA molecules and tested their interactions with purified DNA-PK(CS). The addition of unpaired single strands to blunt DNA ends increased binding and activation of the kinase. When single-stranded loops were added to the DNA ends, binding was preserved, but kinase activation was severely reduced. Obstruction of DNA ends by streptavidin reduced both binding and activation of the kinase. Significantly, short single-stranded oligonucleotides of 3-10 bases were capable of activating DNA-PK(CS). Taken together, these data indicate that kinase activation involves a specific interaction with free single-stranded DNA ends. The structure of DNA-PK(CS) contains an open channel large enough for double-stranded DNA and an adjacent enclosed cavity with the dimensions of single-stranded DNA. The data presented here support a model in which duplex DNA binds to the open channel, and a single-stranded DNA end is inserted into the enclosed cavity to activate the kinase.     

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.     

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.     

Locking of 3' ends of single-stranded DNA templates for improved Pyrosequencing performance

In Pyrosequencing, a DNA strand complementary to a single-stranded DNA (ssDNA) template is synthesized, whereby each incorporated nucleotide yields detectable light, and the light intensity is proportional to the incorporated nucleotides. Correct data interpretation (i.e., signal-to-noise ratio of light intensities) is hampered by artifacts due to the formation of secondary structures of single-stranded templates. Critical among these is the looping back of the template's nonbiotinylated 3' end to itself In the resulting structure, the 3' end functions as a primer, the extension of which results in background signals. We present two ways of preventing the self-priming of a template's 3' end: (i) the use of a modified oligonucleotide, called blOligo, which is complementary to the template's 3' end and (ii) the extension of the template's 3' end with a ddNMP. In contrast to unprotected 3' ends of ssDNA templates, causing inconsistent results, we show that protecting the 3' end of an ssDNA template using either blOligos or ddNMP enables the correct interpretation of signals and results in reliable quantification.     

Assembly and disassembly of RecA protein filaments occur at opposite filament ends. Relationship to DNA strand exchange

RecA protein primarily associates with and dissociates from opposite ends of nucleoprotein filaments formed on linear duplex DNA. RecA nucleoprotein filaments that are hydrolyzing ATP therefore engage in a dynamic process under some conditions that has some of the properties of treadmilling. We have also investigated whether the net polymerization of recA protein at one end of the filament and/or a net depolymerization at the other end drives unidirectional strand exchange. There is no demonstrable correlation between recA protein association/dissociation and the strand exchange reaction. RecA protein-mediated DNA strand exchange is affected minimally by changes in reaction conditions (dilution, pH shift, or addition of small amounts of adenosine-5'-O-(3-thiotriphosphate) that have large and demonstrable effects on recA protein association, dissociation, or both. Rather than driving strand exchange, these assembly and disassembly processes may simply represent the mechanism by which recA nucleoprotein filaments are recycled in the cell.     

Dynamics of RecA filaments on single-stranded DNA

RecA, the key protein in homologous recombination, performs its actions as a helical filament on single-stranded DNA (ssDNA). ATP hydrolysis makes the RecA–ssDNA filament dynamic and is essential for successful recombination. RecA has been studied extensively by single-molecule techniques on double-stranded DNA (dsDNA). Here we directly probe the structure and kinetics of RecA interaction with its biologically most relevant substrate, long ssDNA molecules. We find that RecA ATPase activity is required for the formation of long continuous filaments on ssDNA. These filaments both nucleate and extend with a multimeric unit as indicated by the Hill coefficient of 5.4 for filament nucleation. Disassembly rates of RecA from ssDNA decrease with applied stretching force, corresponding to a mechanism where protein-induced stretching of the ssDNA aids in the disassembly. Finally, we show that RecA–ssDNA filaments can reversibly interconvert between an extended, ATP-bound, and a compressed, ADP-bound state. Taken together, our results demonstrate that ATP hydrolysis has a major influence on the structure and state of RecA filaments on ssDNA.     

Rapid amplification of 5' complementary DNA ends (5' RACE)

This method is used to extend partial cDNA clones by amplifying the 5' sequences of the corresponding mRNAs 1-3. The technique requires knowledge of only a small region of sequence within the partial cDNA clone. During PCR, the thermostable DNA polymerase is directed to the appropriate target RNA by a single primer derived from the region of known sequence; the second primer required for PCR is complementary to a general feature of the target-in the case of 5' RACE, to a homopolymeric tail added (via terminal transferase) to the 3' termini of cDNAs transcribed from a preparation of mRNA. This synthetic tail provides a primer-binding site upstream of the unknown 5' sequence of the target mRNA. The products of the amplification reaction are cloned into a plasmid vector for sequencing and subsequent manipulation.     

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.     

The distribution of Escherichia coli recA protein bound to duplex DNA with single-stranded ends

A short single-stranded tail on one end of an otherwise duplex DNA molecule enables recA protein, in the presence of ATP and MgCl2, to form a complex with the DNA which extends into the duplex portion of the molecule. Nuclease protection studies at a concentration of MgCl2 which permits homologous pairing showed that cleavage by restriction endonucleases at sites throughout the duplex region was inhibited, whereas digestion by DNase I was not affected. These results indicate that recA protein binds to the duplex portion of tailed DNA allowing access by DNase I to a random sample of the many sites at which it cleaves, but providing limited protection of the relatively rare restriction sites. Electron microscopy revealed that the recA nucleoprotein complex with duplex DNA is indeed a segmented or interrupted filament that, with time, extends further from the single-stranded tail into the duplex region. recA protein binding extended into the duplex region more rapidly for duplexes with 5' tails than for those with 3' tails. These observations show that recA protein translocates from a single-stranded region into duplex DNA in the form of a segmented filament by a mechanism that is not strongly polarized.     

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 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.     

Biochemical and biological function of Escherichia coli RecA protein: behavior of mutant RecA proteins

The recA protein of E coli participates in several diverse biological processes and promotes a variety of complex in vitro reactions. A careful comparison of the phenotypic behavior of E coli recA mutations to the biochemical properties of the corresponding mutant proteins reveals a close parallel both between recombination phenotype and DNA strand exchange and renaturation activities, and between inducible phenomena and repressor cleavage activity. The biochemical alterations manifest by the mutant recA proteins are reflected in the strength of their interaction with ssDNA. The defective mutant recA proteins fail to properly assume the high-affinity DNA-binding state that is characteristic of the wild-type protein and, consequently, form less stable complexes with DNA. The mutant proteins displaying an 'enhanced' activity bind ssDNA with approximately the same affinity as the wild-type protein but, due to altered protein-protein interactions, they associate more rapidly with ssDNA. These changes proportionately affect the ability of recA protein to compete with SSB protein, to interact with dsDNA, and, perhaps, to bind repressor proteins. In turn, the DNA strand exchange, DNA renaturation, and repressor cleavage activities mirror these modifications.     

The function of the secondary DNA-binding site of RecA protein during DNA strand exchange.

RecA protein features two distinct DNA-binding sites. During DNA strand exchange, the primary site binds to single-stranded DNA (ssDNA), forming the helical RecA nucleoprotein filament. The weaker secondary site binds double-stranded DNA (dsDNA) during the homology search process. Here we demonstrate that this site has a second important function. It binds the ssDNA strand that is displaced from homologous duplex DNA during DNA strand exchange, stabilizing the initial heteroduplex DNA product. Although the high affinity of the secondary site for ssDNA is essential for DNA strand exchange, it renders DNA strand exchange sensitive to an excess of ssDNA which competes with dsDNA for binding. We further demonstrate that single-stranded DNA-binding protein can sequester ssDNA, preventing its binding to the secondary site and thereby assisting at two levels: it averts the inhibition caused by an excess of ssDNA and prevents the reversal of DNA strand exchange by removing the displaced strand from the secondary site.     

Three-dimensional structure of complexes of single-stranded DNA-binding proteins with DNA. IKe and fd gene 5 proteins form left-handed helices with single-stranded DNA

Specimen-tilting in an electron microscope was used to determine the three-dimensional architecture of the helical complexes formed with DNA by the closely related single-stranded DNA binding proteins of fd and IKe filamentous viruses. The fd gene 5 protein is the only member of the DNA-helix-destabilizing class of proteins whose structure has been determined crystallographically, and yet a parameter essential to molecular modeling of the co-operative interaction of this protein with DNA, the helix handedness, has not been available prior to this work. We find that complexes formed by titrating fd viral DNA with either the fd or IKe gene 5 protein have a left-handed helical sense. Complexes isolated from Escherichia coli infected by fd virus are also found to be left-handed helical; hence, the left-handed fd helices are not an artefact of reconstitution in vitro. Because the proteins and nucleic acid of the complexes are composed of asymmetric units which cannot be fitted equivalently to right-handed and left-handed helices, these results rule out a previous computer graphics atomic model for the helical fd complexes: a right-handed helix had been assumed for the model. Our work provides a defined three-dimensional structural framework within which to model the protein-DNA and protein-protein interactions of two structurally related proteins that bind contiguously and co-operatively on single-stranded DNAs.     

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.