The mutant RecA proteins, RecAR243Q and RecAK245N, exhibit defective DNA binding in homologous pairing

In homologous pairing, the RecA protein sequentially binds to single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA), aligning the two DNA molecules within the helical nucleoprotein filament. To identify the DNA binding region, which stretches from the outside to the inside of the filament, we constructed two mutant RecA proteins, RecAR243Q and RecAK245N, with the amino acid substitutions of Arg243 to Gln and Lys245 to Asn, respectively. These amino acids are exposed to the solvent in the crystal structure of the RecA protein and are located in the central domain, which is believed to be the catalytic center of the homologous pairing activity. The mutations of Arg243 to Gln (RecAR243Q) and Lys245 to Asn (RecAK245N) impair the repair of UV-damaged DNA in vivo and cause defective homologous pairing of ssDNA and dsDNA in vitro. Although RecAR243Q is only slightly defective and RecAK245N is completely proficient in ssDNA binding to form the presynaptic filament, both mutant RecA proteins are defective in the formation of the three-component complex including ssDNA, dsDNA, and RecA protein. The ability to form dsDNA from complementary single strands is also defective in both RecAR243Q and RecAK245N. These results suggest that the region including Arg243 and Lys245 may be involved in the path of secondary DNA binding to the presynaptic filament.     

ATP hydrolysis and the displaced strand are two factors that determine the polarity of RecA-promoted DNA strand exchange.

When the recA protein (RecA) of Escherichia coli promotes strand exchange between single-stranded DNA (ssDNA) circles and linear double-stranded DNAs (dsDNA) with complementary 5' or 3' ends a polarity is observed. This property of RecA depends on ATP hydrolysis and the ssDNA that is displaced in the reaction since no polarity is observed in the presence of the non-hydrolyzable ATP analog, ATP gamma S, or in the presence of single-strand specific exonucleases. Based on these results a model is presented in which both the 5' and 3' complementary ends of the linear dsDNA initiate pairing with the ssDNA circle but only one end remains stably paired. According to this model, the association/dissociation of RecA in the 5' to 3' direction on the displaced strand determines the polarity of strand exchange by favoring or blocking its reinvasion into the newly formed dsDNA. Reinvasion is favored when the displaced strand is coated with RecA whereas it is blocked when it lacks RecA, remains covered by single-stranded DNA binding protein or is removed by a single-strand specific exonuclease. The requirement for ATP hydrolysis is explained if the binding of RecA to the displaced strand occurs via the dissociation and/or transfer of RecA, two functions that depend on ATP hydrolysis. The energy for strand exchange derives from the higher binding constant of RecA for the newly formed dsDNA as compared with that for ssDNA and not from ATP hydrolysis.     

Specific defects in double-stranded DNA unwinding and homologous pairing of a mutant RecA protein

The DNA molecules bound to RecA filaments are extended 1.5-fold relative to B-form DNA. This extended DNA structure may be important in the recognition of homology between single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA). In this study, we show that the K286N mutation specifically impaired the dsDNA unwinding and homologous pairing activities of RecA, without an apparent effect on dsDNA binding itself. In contrast, the R243Q mutation caused defective dsDNA unwinding, due to the defective dsDNA binding of the C-terminal domain of RecA. These results provide new evidence that dsDNA unwinding is essential to homology recognition between ssDNA and dsDNA during homologous pairing.     

Structural and functional analyses of five conserved positively charged residues in the L1 and N-terminal DNA binding motifs of archaeal RADA protein

RecA family proteins engage in an ATP-dependent DNA strand exchange reaction that includes a ssDNA nucleoprotein helical filament and a homologous dsDNA sequence. In spite of more than 20 years of efforts, the molecular mechanism of homology pairing and strand exchange is still not fully understood. Here we report a crystal structure of Sulfolobus solfataricus RadA overwound right-handed filament with three monomers per helical pitch. This structure reveals conformational details of the first ssDNA binding disordered loop (denoted L1 motif) and the dsDNA binding N-terminal domain (NTD). L1 and NTD together form an outwardly open palm structure on the outer surface of the helical filament. Inside this palm structure, five conserved basic amino acid residues (K27, K60, R117, R223 and R229) surround a 25 A pocket that is wide enough to accommodate anionic ssDNA, dsDNA or both. Biochemical analyses demonstrate that these five positively charged residues are essential for DNA binding and for RadA-catalyzed D-loop formation. We suggest that the overwound right-handed RadA filament represents a functional conformation in the homology search and pairing reaction. A new structural model is proposed for the homologous interactions between a RadA-ssDNA nucleoprotein filament and its dsDNA target.     

Changes in the tension in dsDNA alter the conformation of RecA bound to dsDNA�CRecA filaments

The RecA protein is an ATPase that mediates recombination via strand exchange. In strand exchange a single-stranded DNA (ssDNA) bound to RecA binding site I in a RecA/ssDNA filament pairs with one strand of a double-stranded DNA (dsDNA) and forms heteroduplex dsDNA in site I if homology is encountered. Long sequences are exchanged in a dynamic process in which initially unbound dsDNA binds to the leading end of a RecA/ssDNA filament, while heteroduplex dsDNA unbinds from the lagging end via ATP hydrolysis. ATP hydrolysis is required to convert the active RecA conformation, which cannot unbind, to the inactive conformation, which can unbind. If dsDNA extension due to RecA binding increases the dsDNA tension, then RecA unbinding must decrease tension. We show that in the presence of ATP hydrolysis decreases in tension induce decreases in length whereas in the absence of hydrolysis, changes in tension have no systematic effect. These results suggest that decreases in force enhance dissociation by promoting transitions from the active to the inactive RecA conformation. In contrast, increases in tension reduce dissociation. Thus, the changes in tension inherent to strand exchange may couple with ATP hydrolysis to increase the directionality and stringency of strand exchange.     

Coordinated Binding of Single-Stranded and Double-Stranded DNA by UvsX Recombinase

Homologous recombination is important for the error-free repair of DNA double-strand breaks and for replication fork restart. Recombinases of the RecA/Rad51 family perform the central catalytic role in this process. UvsX recombinase is the RecA/Rad51 ortholog of bacteriophage T4. UvsX and other recombinases form presynaptic filaments on ssDNA that are activated to search for homology in dsDNA and to perform DNA strand exchange. To effectively initiate recombination, UvsX must find and bind to ssDNA within an excess of dsDNA. Here we examine the binding of UvsX to ssDNA and dsDNA in the presence and absence of nucleotide cofactor, ATP. We also examine how the binding of one DNA substrate is affected by simultaneous binding of the other to determine how UvsX might selectively assemble on ssDNA. We show that the two DNA binding sites of UvsX are regulated by the nucleotide cofactor ATP and are coordinated with each other such that in the presence of ssDNA, dsDNA binding is significantly reduced and correlated with its homology to the ssDNA bound to the enzyme. UvsX has high affinity for dsDNA in the absence of ssDNA, which may allow for sequestration of the enzyme in an inactive form prior to ssDNA generation.     

Hyper-recombinogenic RecA protein from Pseudomonas aeruginosa with enhanced activity of its primary DNA binding site

According to one prominent model, each protomer in the activated nucleoprotein filament of homologous recombinase RecA possesses two DNA-binding sites. The primary site binds (1) single-stranded DNA (ssDNA) to form presynaptic complex and (2) the newly formed double-stranded (ds) DNA whereas the secondary site binds (1) dsDNA of a partner to initiate strand exchange and (2) the displaced ssDNA following the strand exchange. RecA protein from Pseudomonas aeruginosa (RecAPa) promotes in Escherichia coli hyper-recombination in an SOS-independent manner. Earlier we revealed that RecAPa rapidly displaces E.coli SSB protein (SSB-Ec) from ssDNA to form presynaptic complex. Here we show that this property (1) is based on increased affinity of ssDNA for the RecAPa primary DNA binding site while the affinity for the secondary site remains similar to that for E.coli RecA, (2) is not specific for SSB-Ec but is also observed for SSB protein from P.aeruginosa that, in turn, predicts a possibility of enhanced recombination repair in this pathogenic bacterium.     

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.     

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

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

Crystal structure of RecA from Deinococcus radiodurans: insights into the structural basis of extreme radioresistance

The resistance of Deinococcus radiodurans (Dr) to extreme doses of ionizing radiation depends on its highly efficient capacity to repair dsDNA breaks. Dr RecA, the key protein in the repair of dsDNA breaks by homologous recombination, promotes DNA strand-exchange by an unprecedented inverse pathway, in which the presynaptic filament is formed on dsDNA instead of ssDNA. In order to gain insight into the remarkable repair capacity of Dr and the novel mechanistic features of its RecA protein, we have determined its X-ray crystal structure in complex with ATPgammaS at 2.5A resolution. Like RecA from Escherichia coli, Dr RecA crystallizes as a helical filament that is closely related to its biologically relevant form, but with a more compressed pitch of 67 A. Although the overall fold of Dr RecA is similar to E.coli RecA, there is a large reorientation of the C-terminal domain, which in E.coli RecA has a site for binding dsDNA. Compared to E.coli RecA, the inner surface along the central axis of the Dr RecA filament has an increased positive electrostatic potential. Unique amino acid residues in Dr RecA cluster around a flexible beta-hairpin that has also been implicated in DNA binding.     

RecFOR and RecOR as distinct RecA loading pathways

The molecular role of the RecF protein in loading RecA protein onto single-stranded DNA (ssDNA)-binding protein-coated ssDNA has been obscured by the facility with which the RecO and RecR proteins alone perform this function. We now show that RecFOR and RecOR define distinct RecA loading functions that operate optimally in different contexts. RecFOR, but not RecOR, is most effective when RecF(R) is bound near an ssDNA/double-stranded (dsDNA) junction. However, RecF(R) has no enhanced binding affinity for such a junction. RecO and RecR proteins are both required under all conditions in which the RecFOR pathway operates. The RecOR pathway is uniquely distinguished by a required interaction between RecO protein and the ssDNA binding protein C terminus. The RecOR pathway is more efficient for RecA loading onto ssDNA when no proximal dsDNA is available. A merger of new and published results leads to a new model for RecFOR function.     

Replication protein A interactions with DNA. 2. Characterization of double-stranded DNA-binding/helix-destabilization activities and the role of the zinc-finger domain in DNA interactions

Human replication protein A (RPA) is a heterotrimeric single-stranded DNA-binding protein that is composed of subunits of 70, 32, and 14 kDa. RPA is required for multiple processes in cellular DNA metabolism. RPA has been reported to (1) bind with high affinity to single-stranded DNA (ssDNA), (2) bind specifically to certain double-stranded DNA (dsDNA) sequences, and (3) have DNA helix-destabilizing ("unwinding") activity. We have characterized both dsDNA binding and helix destabilization. The affinity of RPA for dsDNA was lower than that of ssDNA and precisely correlated with the melting temperature of the DNA fragment. The rates of helix destabilization and dsDNA binding were similar, and both were slow relative to the rate of binding ssDNA. We have previously mapped the regions required for ssDNA binding [Walther et al. (1999) Biochemistry 38, 3963-3973]. Here, we show that both helix-destabilization and dsDNA-binding activities map to the central DNA-binding domain of the 70-kDa subunit and that other domains of RPA are needed for optimal activity. We conclude that all types of RPA binding are manifestations of RPA ssDNA-binding activity and that dsDNA binding occurs when RPA destabilizes a region of dsDNA and binds to the resulting ssDNA. The 70-kDa subunit of all RPA homologues contains a highly conserved putative (C-X2-C-X13-C-X2-C) zinc finger. This motif directly interacts with DNA and contributes to dsDNA-binding/unwinding activity. Evidence is presented that a metal ion is required for the function of the zinc-finger motif.     

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

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

Biochemical basis of the constitutive coprotease activity of RecA P67W protein

The mutation of Pro67 to Trp (P67W) in the Escherichia coli RecA protein results in reduced recombination and constitutive coprotease phenotypes. We examined the biochemical properties of this mutant in an effort to understand these altered behaviors. We find that RecA P67W protein can access single-stranded DNA (ssDNA) binding sites within regions of secondary structure more effectively than wild-type protein, and binding to duplex DNA is both faster and more extensive as well. This mutant is also more effective than wild-type RecA protein in displacing SSB protein from ssDNA. An enhancement in SSB protein displacement has been shown previously for RecA441, RecA730, and RecA803 proteins, and similarly, this improved ability to displace SSB protein for RecA P67W protein correlates with an increased rate of association with ssDNA. As for the aforementioned mutant RecA proteins, we expect that this enhanced activity will allow RecA P67W protein to bind ssDNA naturally occurring in undamaged cells and to constitutively induce the SOS response. The DNA strand exchange activity of RecA P67W protein is also altered. Although the rate of duplex DNA uptake into joint molecules is increased compared to that of wild-type RecA protein, the resolution to the nicked circular dsDNA product is reduced. We suggest that either a limited amount of DNA strand reinvasion or a defect in DNA heteroduplex extension is responsible for the impaired recombination ability of this mutant protein.     

Electron microscopic visualization of the RecA protein-mediated pairing and branch migration phases of DNA strand exchange

The RecA protein of Escherichia coli will drive the pairing and exchange of strands between homologous DNA molecules in a reaction stimulated by single-stranded binding protein. Here, reactions utilizing three homologous DNA pairs which can undergo both paranemic and plectonemic joining were examined by electron microscopy: supertwisted double-stranded (ds) DNA and linear single-stranded (ss) DNA, linear dsDNA and circular ssDNA, and linear dsDNA and colinear ssDNA. Several major observations were: (i) with RecA protein bound to the DNA, plectonemic joints were ultrastructurally indistinguishable from paranemic joints; (ii) complexes which appeared to be joined both paranemically and plectonemically were present in these reactions in roughly equal numbers; and (iii) in complexes undergoing strand exchange, both DNA partners were often enveloped within a RecA protein filament consisting of hundreds of RecA protein monomers and several kilobases of DNA. These observations suggest that, following RecA protein-ssDNA filament formation, strand exchange proceeds by a pathway that can be divided structurally into three phases: pairing, envelopment/exchange, and release of the products.     

The homologous pairing domain of RecA also mediates the allosteric regulation of DNA binding and ATP hydrolysis: a remarkable concentration of functional residues

Switching between the active (ATP and DNA bound) and inactive conformations of the homologous recombination RecA protein is regulated by ATP hydrolysis. First, we use the homologous pairing domain of RecA derived from its mobile loop L2 to show that the interaction of this random coil peptide with the gamma-phosphate of ATP results in a peptide beta-conformation similar to that previously shown to be induced by DNA binding. Next, we show that in the whole RecA protein two residues in this L2 domain, Gln194 and Arg196, are catalytic amino acid residues for ATP hydrolysis and functionally resemble the corresponding residues engaged in GTP hydrolysis by two distinct classes of G proteins. Finally, we show that the role of DNA and high salt in the stimulation of the ATPase of RecA is to stabilize this highly mobile region involved in hydrolysis. This is a role similar to that described for RGSs in the activation of the GTPase of heterotrimeric G proteins. Therefore, (i) a prototypical DNA-dependent ATPase and ATP-stimulated DNA-binding protein, RecA, and eukaryotic signaling proteins share common stereochemical regulatory mechanisms; and (ii) in a remarkable example of parsimony, loop L2 is a molecular switch that controls both ATP promoted DNA binding and pairing reactions and DNA stimulated ATP hydrolysis.     

Modeling the early stage of DNA sequence recognition within RecA nucleoprotein filaments

Homologous recombination is a fundamental process enabling the repair of double-strand breaks with a high degree of fidelity. In prokaryotes, it is carried out by RecA nucleofilaments formed on single-stranded DNA (ssDNA). These filaments incorporate genomic sequences that are homologous to the ssDNA and exchange the homologous strands. Due to the highly dynamic character of this process and its rapid propagation along the filament, the sequence recognition and strand exchange mechanism remains unknown at the structural level. The recently published structure of the RecA/DNA filament active for recombination (Chen et al., Mechanism of homologous recombination from the RecA-ssDNA/dsDNA structure, Nature 2008, 453, 489) provides a starting point for new exploration of the system. Here, we investigate the possible geometries of association of the early encounter complex between RecA/ssDNA filament and double-stranded DNA (dsDNA). Due to the huge size of the system and its dense packing, we use a reduced representation for protein and DNA together with state-of-the-art molecular modeling methods, including systematic docking and virtual reality simulations. The results indicate that it is possible for the double-stranded DNA to access the RecA-bound ssDNA while initially retaining its Watson–Crick pairing. They emphasize the importance of RecA L2 loop mobility for both recognition and strand exchange.     

An alpha-helical peptide model for electrostatic interactions of proteins with DNA. The N terminus of RecA

A series of synthetic peptides have been studied as models for non-specific protein-DNA interactions. In an alpha-helical conformation, the charged amino acid residues of the N-terminal 24 residues of RecA protein are asymmetrically distributed; at neutral pH there is a +4 charge on one face of the helix and a -3 charge on the other face. Modeling suggests that the positive face of the helix can bind five DNA phosphate groups by electrostatic interactions. Circular dichroism (c.d.) spectra indicate that the analogous peptide, Rec24 (AIDENKQKALAAALGQIEKQFGKG-amide), is largely unstructured in water but becomes highly helical in the presence of DNA. Peptide titrations of fluorescent etheno-DNA confirm that the changes in the c.d. spectrum of the peptide are associated with binding, although a dependence of the c.d. signal on the degree of DNA saturation is observed, indicating that peptide can be bound in more than one conformation. At saturation the peptide binds to 5.0(+/- 0.5) DNA phosphate groups as predicted and the electrostatic nature of the binding is confirmed by a strong dependence on salt concentration. A "mutant" peptide where an acidic glutamate residue replaces an alanine on the basic face of the Rec24 helix exhibits weaker binding to single-stranded DNA, also consistent with the electrostatic nature of the proposed peptide-DNA interaction. Extending Rec24 by ten amino acid residues, where the additional residues do not participate in the helical motif, does not noticeably affect binding. Thus, we show experimentally that an asymmetric charge distribution on an alpha-helix can represent an important element for binding nucleic acids.     

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.     

A Phage Single-Stranded DNA (ssDNA) Binding Protein Complements ssDNA Accumulation of a Geminivirus and Interferes with Viral Movement

Geminiviruses are plant viruses with circular single-stranded DNA (ssDNA) genomes encapsidated in double icosahedral particles. Tomato leaf curl geminivirus (ToLCV) requires coat protein (CP) for the accumulation of ssDNA in protoplasts and in plants but not for systemic infection and symptom development in plants. In the absence of CP, infected protoplasts accumulate reduced levels of ssDNA and increased amounts of double-stranded DNA (dsDNA), compared to accumulation in the presence of wild-type virus. To determine whether the gene 5 protein (g5p), a ssDNA binding protein from Escherichia coli phage M13, could restore the accumulation of ssDNA, ToLCV that lacked the CP gene was modified to express g5p or g5p fused to the N-terminal 66 amino acids of CP (CP66:6G:g5). The modified viruses led to the accumulation of wild-type levels of ssDNA and high levels of dsDNA. The accumulation of ssDNA was apparently due to stable binding of g5p to viral ssDNA. The high levels of dsDNA accumulation during infections with the modified viruses suggested a direct role for CP in viral DNA replication. ToLCV that produced the CP66:6G:g5 protein did not spread efficiently in Nicotiana benthamiana plants, and inoculated plants developed only very mild symptoms. In infected protoplasts, the CP66:6G:g5 protein was immunolocalized to nuclei. We propose that the fusion protein interferes with the function of the BV1 movement protein and thereby prevents spread of the infection.