Defective homologous pairing and proficient processive unwinding by the recA430 mutant protein and intermediates of homologous pairing by recA protein

The recA protein promotes the formation and processing of joint molecules of homologous double- and single-stranded DNAs in vitro. Under a set of specified conditions, we found that the substitution of a single amino acid in the recA protein (recA430 mutation) depresses its activity for the homologous pairing to about 1/100 of that by the wild type protein when compared by the rate for the first 2-3 min of the reaction, but that the mutation only slightly, if at all, affects its ability to bind progressively to double-stranded DNA to unwind the double helix ("processive unwinding"). This is in striking contrast to an anti-recA protein monoclonal IgG, ARM193, which severely inhibits the processive unwinding but not the homologous pairing, providing further support for our conclusion that the homologous pairing and processive unwinding are functionally independent of each other. Antibody ARM193 caused the breakdown of spontaneously formed filaments of the recA protein, but the recA430 mutation did not affect the self-polymerization of the protein. The recA430 protein was apparently proficient in the functional binding to a single-stranded DNA and in the hydrolysis of ATP. However, we found that under the above conditions the mutant protein was defective as to homology-independent conjunction of DNA molecules to form a "ternary complex" (of macromolecules). These results suggest that (i) only one DNA-binding site is sufficient for the recA protein to promote the processive unwinding (the ability of the protein to form spontaneous filaments is closely related to this process) and that (ii) two DNA-binding sites on each of the recA polypeptides or those composed of a dimer (or oligomer) of the polypeptide are required for the recA protein to promote both the conjunction of parental DNA molecules and the homologous pairing (the ability to form the spontaneous filaments is not essential to this process). (iii) The simultaneous inactivation of the activity to promote the homologous pairing and that to form the ternary complex by the single substitution of the amino acid provides a physical support for the conclusion that the ternary complex is an indispensable intermediate in the homologous pairing.     

ATP-dependent unwinding of the double helix and extensive supercoiling by Escherichia coli recA protein in the presence of topoisomerase

recA protein, which is essential for genetic recombination in Escherichia coli, causes extensive unwinding of the double helix by an ATP-dependent reaction and accumulation of positive supercoiling in closed circular double-stranded DNA. Initiation of the extensive unwinding was largely dependent on homologous single-stranded DNA. Therefore, it is likely that the extensive unwinding is initiated mainly at the site of D-loops. "Nascent D-loops" in which the two DNA molecules did not interwind were also good initiation sites of extensive unwinding. When the concentration of Mg2+ was decreased from the standard conditions for D-loop formation (13 mM MgCl2; the higher Mg2+ condition) to the lower Mg2+ condition (1 to 2 mM MgCl2), extensive unwinding by recA protein was initiated very quickly in the absence of single-stranded DNA. Results showed that this single-stranded DNA-independent initiation of extensive unwinding (i) requires negative superhelicity of the double-stranded DNA and (ii) is a first order reaction with respect to the DNA. These observations suggest that, under the lower Mg2+ condition, the extensive unwinding starts at a transiently denatured site in the negative superhelical DNA. Once initiated, the unwinding by recA protein is propagated extensively, even under conditions that do not allow its initiation. Therefore, the propagation of unwinding is a processive reaction ("processive unwinding"). Previous studies indicated that recA protein promotes "distributive unwinding" of double helix which depends on single-stranded DNA. Therefore, recA protein promotes unwinding of the double helix by either of two distinct pathways. Stress caused by the processive unwinding could explain the dissociation of D-loops and reversible inactivation of the double-stranded DNA in a D-loop cycle.     

Epitopes and active sites of the RecA protein.

The RecA protein is indispensable for homologous genetic recombination in Escherichia coli. This protein alone promotes the ATP-dependent formation of homologous joint molecules and their processing in vitro. Through the use of a set of anti-RecA protein mouse monoclonal IgGs, we have been attempting to divide the whole process into elementary steps to determine the basic functions of the protein. In order to correlate the basic functions with the active sites on the recA polypeptide, we located the epitopes for the anti-RecA protein-IgGs on the recA polypeptide by means of immunoblotting experiments and an enzyme-linked immunosorbent assay involving isolated proteolytic polypeptides or synthetic ones derived from various regions of the recA polypeptide. The epitopes for anti-RecA protein-IgGs ARM321 and ARM414, both of which are shown to inhibit the DNA-dependent ATP hydrolysis and the formation of homologous joints by the RecA protein, were found to be located between Thr89 and Glu127 and between Glu233 and Lys256, respectively, on the RecA polypeptide. IgG ARM193 had been shown to interfere with the protein-protein interaction between two RecA protein molecules, and ARM191 had been suggested to inhibit the binding of double-stranded DNA to the RecA protein. The epitopes for ARM193 and ARM191 were found to be located in a approximately 90-amino acid region at the C terminus. These results suggest the locations of the active sites and a functional core on the RecA polypeptide.     

Formation of heteroduplex DNA promoted by the combined activities of Escherichia coli recA and recBCD proteins

We have established an in vitro reaction in which heteroduplex DNA formation is dependent on the concerted actions of recA and recBCD proteins, the major components of the recBCD pathway of genetic recombination in vivo. We find that heteroduplex DNA formation requires three distinct enzymatic functions: first, the helicase activity of recBCD enzyme initiates heteroduplex DNA formation by unwinding the linear double-stranded DNA molecule to transiently form single-stranded DNA (ssDNA); second, recA protein traps this ssDNA before it reanneals; third, recA protein catalyzes the pairing of this ssDNA molecule with another homologous ssDNA molecule, followed by the renaturation of these molecules to form heteroduplex DNA. The first two functions should be important to all in vitro reactions involving recA and recBCD proteins, whereas the third will be specific to the DNA substrates used. The rate-limiting step of heteroduplex DNA formation is the trapping by recA protein of the ssDNA produced by recBCD enzyme. A model for this reaction is described.     

Networks of DNA and RecA protein are intermediates in homologous pairing

Partial coating of single-stranded DNA by recA protein causes its aggregation, but conditions that promote complete coating inhibit independent aggregation of single strands and, instead, cause the mutually dependent conjunction of single- and double-stranded DNA in complexes that sediment at more than 10 000 S. This coaggregation is independent of homology but otherwise shares key properties of homologous pairing of single strands with duplex DNA: both processes require ATP, MgCl2, and stoichiometric amounts of recA protein; both are very sensitive to inhibition by salt and ADP. Coaggregates are closed domains that are intermediates in homologous pairing: they form faster than joint molecules, they include virtually all of the DNA in the reaction mixture, and they yield joint molecules nearly an order of magnitude faster than they exchange DNA molecules with the surrounding solution. The independent aggregation of single-stranded DNA differs in all respects except the requirement for Mg2+, and its properties correlate instead with those associated with the renaturation of complementary single strands by recA protein.     

Properties of recA441 protein-catalyzed DNA strand exchange can be attributed to an enhanced ability to compete with SSB protein

We have investigated the recombinase activity of recA441 protein by comparing its in vitro DNA strand exchange activity to that of wild-type recA protein. Consistent with its proficiency in recombination in vivo, recA441 protein is able to catalyze the in vitro exchange of a circular single-stranded DNA molecule for a homologous strand in a linear double-stranded DNA molecule. Under conditions optimal for wild-type recA protein, the rates of joint molecule formation are the same for the two recA proteins, but the wild-type protein converts these intermediate species to gapped circular heteroduplex DNA product molecules more rapidly than recA441 protein. In the recA441 protein reaction, joint molecules are instead converted to extensive homology-dependent DNA networks via presumed reinitiation reactions. Under some conditions, the DNA strand exchange activity of recA441 protein is enhanced relative to the wild-type. These conditions include when single-stranded DNA.SSB protein (where SSB is Escherichia coli single-stranded DNA-binding protein) complexes are formed prior to the addition of recA protein, at low magnesium ion concentration in the presence of spermidine, and at low ATP concentrations. Under the conditions examined, recA441 protein competes more effectively with SSB protein for DNA-binding sites; thus, the differences between the strand exchange activities of the wild-type and recA441 proteins can be attributed to this enhanced ability in SSB protein competition.     

On the mechanism of pairing of single- and double-stranded DNA molecules by the recA and single-stranded DNA-binding proteins of Escherichia coli

The pairing of single- and double-stranded DNA molecules at homologous sequences promoted by recA and single-stranded DNA-binding proteins of Escherichia coli follows apparent first-order kinetics. The initial rate and first-order rate constant for the reaction are maximal at approximately 1 recA protein/3 and 1 single-stranded DNA-binding protein/8 nucleotides of single-stranded DNA. The initial rate increases with the concentration of duplex DNA; however, the rate constant is independent of duplex DNA concentration. Both the rate constant and extent of reaction increase linearly with increasing length of duplex DNA over the range 366 to 8623 base pairs. In contrast, the rate constant is independent of the size of the circular single-stranded DNA between 6,400 and 10,100 nucleotides. No significant effect on reaction rate is observed when a single-stranded DNA is paired with 477 base pairs of homologous duplex DNA joined to increasing lengths of heterologous DNA (627-2,367 base pairs). Similarly, heterologous T7 DNA has no effect on the rate of pairing. These findings support a mechanism in which a recA protein-single-stranded DNA complex interacts with the duplex DNA to produce an intermediate in which the two DNA molecules are aligned at homologous sequences. Conversion of the intermediate to a paranemic joint then occurs in a rate-determining unimolecular process.     

Homologous pairing in genetic recombination: recA protein makes joint molecules of gapped circular DNA and closed circular DNA

The recA protein, which is essential for genetic recombination in E. coli, promotes the homologous pairing of double-stranded DNA and linear single-stranded DNA, thereby forming a three-stranded joint molecule called a D loop. Single-stranded DNA stimulates recA protein to unwind double-stranded DNA. By a presumably related mechanism, recA protein promoted the homologous pairing of two circular double-stranded molecules when one of them had a gap in one strand. The two molecules were joined at homologous sites by noncovalent bonds. The covalently closed molecule remained intact and was not topologically linked to the intact circular strand of the gapped substrate. Electron microscopy showed that molecules were usually linked at two or more nearby points. The junctions in most molecules were shorter than 300 nucleotides. Sometimes the region between two extreme points was separated into two arms, producing an ellipsoidal loop (called an eye loop). The junctions in these biparental joint molecules were frequently remote from the site of the gap. We infer that a free end of the interrupted strand crossed over to form a structure like a D loop which moved away from the gap by branch migration.     

Intermediates in homologous pairing promoted by recA protein. Isolation and characterization of active presynaptic complexes

recA protein promotes homologous pairing and strand exchange by an ordered reaction in which the protein first polymerizes on single-stranded DNA. This presynaptic intermediate, which can be formed either in the presence or absence of Escherichia coli single-stranded binding protein (SSB), has been isolated by gel filtration and characterized. At saturation, purified complexes contained one molecule of recA protein per 3.6 nucleotide residues of single-stranded DNA. Complexes that had been formed in the presence of SSB contained up to one molecule of SSB per 15 nucleotide residues, but the content of SSB in different preparations of isolated complexes appeared to be inversely related to the content of recA protein. Even when they have lost as much as a third of their recA protein, presynaptic complexes can retain activity, because the formation of stable joint molecules depends principally on the binding of recA protein to the single-stranded DNA in the localized region that corresponds to the end of the duplex substrate.     

Increase of the DNA strand assimilation activity of recA protein by removal of the C terminus and structure-function studies of the resulting protein fragment

A proteolytic fragment of recA protein, missing about 15% of the protein at the C terminus, was found to promote assimilation of homologous single-stranded DNA into duplex DNA more efficiently than intact recA protein. This difference was not found if Escherichia coli single-stranded DNA binding protein was present. The ATPase activity of both intact recA protein and the fragment was identical. The difference in strand assimilation activity cannot be due to differences in single-stranded DNA affinity, since both the fragment and intact proteins bind to single-stranded DNA with nearly identical affinities. However, the fragment was found to bind double-stranded DNA more tightly and to aggregate more extensively than recA protein; both of these properties may be important in strand assimilation. Aggregation of the fragment was extensive in the presence of duplex DNA under the same condition where recA protein did not aggregate. The double-stranded DNA binding of both recA protein and the fragment responds to nucleotide cofactors in the same manner as single-stranded DNA binding, i.e. ADP weakens and ATP gamma S strengthens the association. The missing C-terminal region of recA protein includes a very acidic region that is homologous to other single-stranded DNA binding proteins and which has been implicated in DNA binding modulation. This C-terminal region may serve a similar function in recA protein, possibly inhibiting double-stranded DNA invasion. The possible role of the enhanced double-stranded DNA affinity of the fragment protein in the mechanism of strand assimilation is discussed.     

T4 phage gene uvsX product catalyzes homologous DNA pairing.

Gene uvsX of phage T4 controls genetic recombination and the repair of DNA damage. We have recently purified the gene product, and here describe its properties. The protein has a single-stranded DNA-dependent ATPase activity. It binds efficiently to single- and double-stranded DNAs at 0 degrees C in a cooperative manner. At 30 degree C the double-stranded DNA-protein complex was stable, but the single-stranded DNA-protein complex dissociated rapidly. The instability of the latter complex was reduced by ATP. The protein renatured heat-denatured double-stranded DNA, and assimilated linear single-stranded DNA into homologous superhelical duplexes to produce D-loops. The reaction is stimulated by gene 32 protein when the uvsX protein is limiting. With linear double-stranded DNA and homologous, circular single-stranded DNA, the protein catalyzed single-strand displacement in the 5' to 3' direction with the cooperation of gene 32 protein. All reactions required Mg2+, and all except DNA binding required ATP. We conclude that the uvsX protein is directly involved in strand exchange and is analogous to the recA protein of Escherichia coli. The differences between the uvsX protein and the recA protein, and the role of gene 32 protein in single-strand assimilation and single-strand displacement are briefly discussed.     

Homologous pairing between nucleosome cores on a linear duplex DNA and nucleoprotein filaments of RecA protein-single stranded DNA.

The ability of E coli recA protein to promote homologous pairing with linear duplex DNA bound to HU protein (Nucleosome cores) was found to be differentially affected. The formation of paranemic joint molecules was not affected whereas the formation of plectomic joint molecules was inhibited from the start of the reaction. The formation of paranemic joint molecules between nucleoprotein filaments of recA protein-circular single stranded DNA and closed circular duplex DNA is believed to generate positive supercoiling in the duplex DNA. We found that the positively superhelical duplex DNA was inert in the formation of joint molecules but could be converted into an active substrate, in situ, by the action of wheat germ topoisomerase I. These observations initiate an understanding of the structural features of E coli chromosome such as DNA supercoiling and nucleosome-like structures in homologous recombination.     

RecA protein promoted homologous pairing in vitro. Pairing between linear duplex DNA bound to HU Protein (nucleosome cores) and nucleoprotein filaments of recA protein-single-stranded DNA

RecA protein promotes two distinct types of synaptic structures between circular single strands and duplex DNA; paranemic joints, where true intertwining of paired strands is prohibited and the classically intertwined plectonemic form of heteroduplex DNA. Paranemic joints are less stable than plectonemic joints and are believed to be the precursors for the formation of plectonemic joints. We present evidence that under strand exchange conditions the binding of HU protein, from Escherichia coli, to duplex DNA differentially affects homologous pairing in vitro. This conclusion is based on the observation that the formation of paranemic joint molecules was not affected, whereas the formation of plectonemic joint molecules was inhibited from the start of the reaction. Furthermore, introduction of HU protein into an ongoing reaction stalls further increase in the rate of the reaction. By contrast, binding of HU protein to circular single strands has neither stimulatory nor inhibitory effect. Since the formation of paranemic joint molecules is believed to generate positive supercoiling in the duplex DNA, we have examined the ability of positive superhelical DNA to serve as a template in the formation of paranemic joint molecules. The inert positively supercoiled DNA could be converted into an active substrate, in situ, by the action of wheat germ topoisomerase I. Taken collectively, these results indicate that the structural features of the bacterial chromosome which include DNA supercoiling and organization of DNA into nucleosome-like structures by HU protein modulate homologous pairing promoted by the nucleoprotein filaments of recA protein single-stranded DNA.     

Homologous pairing of single-stranded DNA and superhelical double-stranded DNA catalyzed by RecO protein from Escherichia coli.

The recO gene product is required for DNA repair and some types of homologous recombination in wild-type Escherichia coli cells. RecO protein has been previously purified and shown to bind to single- and double-stranded DNA and to promote the renaturation of complementary single-stranded DNA molecules. In this study, purified RecO protein was shown to catalyze the assimilation of single-stranded DNA into homologous superhelical double-stranded DNA, an activity also associated with RecA protein. The RecO protein-promoted strand assimilation reaction requires Mg2+ and is ATP independent. Because of the biochemical similarities between RecO and RecA proteins, the ability of RecO protein to substitute for RecA protein in DNA repair in vivo was also assessed in this study. The results show that overexpression of RecO protein partially suppressed the UV repair deficiency of a recA null mutant and support the hypothesis that RecO and RecA proteins are functionally similar with respect to strand assimilation and the ability to enhance UV survival. These results suggest that RecO and RecA proteins may have common functional properties.     

Recombination hotspots and single-stranded DNA binding proteins couple DNA translocation to DNA unwinding by the AddAB helicase-nuclease

AddAB is a helicase-nuclease that processes double-stranded DNA breaks for repair by homologous recombination. This process is modulated by Chi recombination hotspots: specific DNA sequences that attenuate the nuclease activity of the translocating AddAB complex to promote downstream recombination. Using a combination of kinetic and imaging techniques, we show that AddAB translocation is not coupled to DNA unwinding in the absence of single-stranded DNA binding proteins because nascent single-stranded DNA immediately re-anneals behind the moving enzyme. However, recognition of recombination hotspot sequences during translocation activates unwinding by coupling these activities, thereby ensuring the downstream formation of single-stranded DNA that is required for RecA-mediated recombinational repair. In addition to their implications for the mechanism of double-stranded DNA break repair, these observations may affect our implementation and interpretation of helicase assays and our understanding of helicase mechanisms in general.     

Homologous pairing in duplex DNA regions and the formation of four-stranded paranemic joints promoted by RecA protein. Effects of gap length and negative superhelicity

RecA protein catalyzes homologous pairing of partially single-stranded duplex DNA and fully duplex DNA to form stable joint molecules. We constructed circular duplex DNA with various defined gap lengths and studied the pairing reaction between the gapped substrate with fully double-stranded DNA. The reaction required a stoichiometric amount of RecA protein, and the optimal reaction was achieved at a ratio of 1 RecA monomer per 4 base pairs. The length of the gap, ranging from 141 to 1158 nucleotides, had little effect on the efficiency of homologous pairing. By using a circular gapped duplex DNA prepared from the chimeric phage M13Gori1, we were able to show the formation of nonintertwined or paranemic joints in duplex regions between the gapped and fully duplex molecules. The formation of such paranemic joints occurred efficiently and included nearly all of the DNA in the reaction mixture. The reaction required negative superhelicity, and pairing was greatly reduced with linear or nicked circular DNA. We conclude that one functional role of the single-stranded gap is for facilitating the binding of RecA protein to the duplex region of the gapped DNA. Once the nucleoprotein filament is formed, homologous pairing between the gapped and fully duplex DNA can take place anywhere along the length of the nucleoprotein complex.     

RecA protein-promoted homologous pairing between duplex molecules: functional role of duplex regions of gapped duplex DNA

RecA protein promotes homologous pairing and symmetrical strand exchange between partially single-stranded duplex DNA and fully duplex molecules. We constructed circular gapped DNA with a defined gap length and studied the pairing reaction between the gapped substrate and fully duplex DNA. RecA protein polymerizes onto the single-stranded and duplex regions of the gapped DNA to form a nucleoprotein filament. The formation of such filaments requires a stoichiometric amount of RecA protein. Both the rate and yield of joint molecule formation were reduced when the pairing reaction was carried out in the presence of a sub-saturating amount of RecA protein. The amount of RecA protein required for optimal pairing corresponds to the binding site size of RecA protein at saturation on duplex DNA. The result suggests that in the 4-stranded system the single-stranded as well as the duplex regions are involved in pairing. By using fully duplex DNA that shares different lengths and regions of homology with the gapped molecule, we directly showed that the duplex region of the gapped DNA increased both the rate and yield of joint molecule formation. The present study indicates that even though strand exchange in the 4-stranded system must require the presence of a single-stranded region, the pairing that occurs in duplex regions between DNA molecules is functionally significant and contributes to the overall activity of the gapped DNA.     

Monoclonal antibodies with specific effects on partial activities of recA protein of Escherichia coli

recA protein of Escherichia coli promotes a wide variety of DNA reactions in vitro. Specific effectors of recA protein should be very useful in elucidating the mechanisms of these complex reactions. Six mouse hybridoma clones that secreted class G immunoglobulins specific to recA protein were obtained in three cell-fusion experiments. Five IgGs were purified by affinity chromatography. These monoclonal antibodies were characterized by examining their effects on the single-stranded DNA-dependent ATPase activity, negatively superhelical double-stranded DNA-dependent ATPase activity, and an activity in pairing negatively superhelical closed circular double-stranded DNA and homologous single-stranded DNA-fragments to form D-loops. These IgGs inhibited all, some, or one of these three activities, and from the spectra of their inhibitory effects they were classified into four groups. This classification suggests that each of the monoclonal antibodies binds to one of at least four antigenic determinants on recA protein and specifically inactivates one or more of the active centers on the protein. These monoclonal antibodies will be useful in analyzing the complex reactions promoted by recA protein.     

Can recA protein promote homologous pairing between duplex regions of DNA?

RecA protein has been shown to promote the formation of joint molecules between intact duplex DNA and homologous gapped DNA. When examined by electron microscopy, such joint molecules display a junction that is, in most cases, distant from the site of the gap. This led us to test whether the observed location of the joint was due to pairing at the gap followed by branch migration, or whether recA-promoted pairing could also take place between duplex homologous regions away from the gap. To test the latter possibility, intact duplex DNA was incubated with DNA which contained a gap in a region of non-homology. Joint molecules were detected by filter binding assay and by electron microscopy at about one-third of the yield observed for fully homologous molecules. These results indicate that initial homologous pairing promoted by recA protein is not restricted to the single-stranded region in the gap but can also take place in regions where both molecules are duplex.     

Synapsis promoted by Ustilago rec1 protein

Ustilago rec 1 protein pairs homologous DNA molecules by promoting both synapsis and strand transfer. Complexes formed with rec 1 protein and a homologous combination of single-stranded and duplex DNA that appear to be synaptic structures can be detected by use of a nitrocellulose filter assay. The nascent heteroduplex formed during synapsis is a paranemic joint in which the single-stranded DNA pairs, but does not interwind, with its complement in the duplex molecule. Formation of the paranemic joint is accompanied by duplex unwinding and genesis of left-handed Z-DNA.