RNA-DNA hybridization promoted by E. coli RecA protein.

RecA protein of E. coli plays a central regulatory role that is induced by damage to DNA and results in the inactivation of LexA repressor. In vitro, RecA protein binds preferentially to single-stranded DNA to form a nucleoprotein filament that can recognize homology in naked duplex DNA and promote extensive strand exchange. Although RecA protein shows little tendency at neutral pH to bind to RNA, we found that it nonetheless catalyzed at 37 degrees C the hybridization of complementary RNA and single-stranded DNA sequences. Hybrids made by RecA protein at 37 degrees C appeared indistinguishable from ones prepared by thermal annealing. RNA-DNA hybridization by RecA protein at neutral pH required, as does RecA-promoted homologous pairing, optimal conditions for the formation of RecA nucleoprotein filaments. The cosedimentation of RNA with those filaments further paralleled observations made on the formation of networks of nucleoprotein filaments with double-stranded DNA, an instrumental intermediate in homologous pairing in vitro. These similarities with the pairing reaction support the view that RecA protein acts specifically in the hybridization reaction.     

Molecular visualization of the yeast Dmc1 protein ring and Dmc1-ssDNA nucleoprotein complex

Saccharomyces cerevisiae Dmc1, a meiosis-specific homologue of RecA, catalyzes homologous pairing and strand exchange during meiotic DNA recombination. The purified budding yeast Dmc1 (ScDmc1) protein exhibits much weaker recombinase activity in vitro as compared to that of the Escherichia coli RecA protein. Using atomic force microscopy (AFM) with carbon nanotube tips, we found ScDmc1 forms rings with an external diameter of 18 nm and a central cavity of 4 nm. In the presence of single-stranded DNA (ssDNA), the majority of the ScDmc1 protein (90%) bound DNA as protein rings; only a small faction (10%) was able to form filamentous structure. In contrast, nearly all RecA proteins form fine helical nucleoprotein filaments with ssDNA under identical conditions. RecA-mediated recombinase activity is initiated through the nucleation of RecA onto ssDNA to form helical nucleoprotein filaments. Our results support the notion that ScDmc1 becomes catalytically active only when it forms a helical nucleoprotein filament with ssDNA.     

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.     

A DNA pairing-enhanced conformation of bacterial RecA proteins

The RecA proteins of Escherichia coli (Ec) and Deinococcus radiodurans (Dr) both promote a DNA strand exchange reaction involving two duplex DNAs. The four-strand exchange reaction promoted by the DrRecA protein is similar to that promoted by EcRecA, except that key parts of the reaction are inhibited by Ec single-stranded DNA-binding protein (SSB). In the absence of SSB, the initiation of strand exchange is greatly enhanced by dsDNA-ssDNA junctions at the ends of DNA gaps. This same trend is seen with the EcRecA protein. The results lead to an expansion of published hypotheses for the pathway for RecA-mediated DNA pairing, in which the slow first order step (observed in several studies) involves a structural transition to a state we designate P. The P state is identical to the state found when RecA is bound to double-stranded (ds) DNA. The structural state present when the RecA protein is bound to single-stranded (ss) DNA is designated A. The DNA pairing model in turn facilitates an articulation of three additional conclusions arising from the present work. 1) When a segment of a RecA filament bound to ssDNA is forced into the P state (as RecA bound to the ssDNA immediately adjacent to dsDNA-ssDNA junction), the segment becomes "pairing enhanced." 2) The unusual DNA pairing properties of the D. radiodurans RecA protein can be explained by postulating this protein has a more stringent requirement to initiate DNA strand exchange from the P state. 3) RecA filaments bound to dsDNA (P state) have directly observable structural changes relative to RecA filaments bound to ssDNA (A state), involving the C-terminal domain.     

Visualization of recA protein and its association with DNA: a priming effect of single-strand-binding protein

A stoichiometric interaction of RecA protein with single-stranded DNA promotes homologous pairing of the single strand with duplex DNA and subsequent polar formation of a heteroduplex joint. Escherichia coli single-strand-binding (SSB) protein augments these reactions. Electron microscopic observations suggest structural bases for these interactions. Without triphosphates or DNA, RecA protein forms short linear filaments. With added circular single-stranded DNA, it forms extended circular filaments as well as collapsed and aggregated complexes of protein and DNA. The extended circular filaments are stiff and regular in appearance, contrasting with the convoluted structure formed by SSB protein and single-stranded DNA. Together, these two proteins form mixed filaments, which mostly resemble the extended structures containing RecA protein; moreover, SSB protein accelerates formation of extended filaments more than 50-fold, increasing the yield of these structures at the expense of heterogeneous aggregates. Other observations further define the interactions of RecA protein with partially single-stranded DNA, and the effects of ATP gamma S on the tendency of RecA protein to form polymeric structures even in the absence of DNA.     

RecA-AC: single-site cleavage of plasmids and chromosomes at any predetermined restriction site.

We have developed a novel version of the Achilles' Cleavage (AC) reaction in which virtually any restriction site on DNA of any size can be converted to a unique cleavage site. We first polymerized RecA protein on a synthetic oligodeoxyribonucleotide (oligo) in the presence of a nonhydrolyzable ATP analogue to generate oligo:RecA nucleoprotein filaments. These filament were then incubated with plasmid or intact chromosomal DNA from Saccharomyces cerevisiae to form stable complexes in the yeast LEU2 gene at the target sequence identical (or complementary) to that of the oligo. When HhaII (HinfI) methyltransferase (M.HhaII) was added, all of the recognition sites for HhaII with the exception of the one protected by the RecA filament were methylated and thus no longer cleaved by the cognate restriction endonuclease (HinfI). After inactivation of the RecA and the M.HhaII, HinfI was used to efficiently cleave the plasmid or chromosome specifically at the targeted restriction site. Since oligos specific for any sequence can be easily synthesized and the other reagents necessary to perform RecA-mediated AC (RecA-AC) reactions on both plasmids and intact chromosomes are readily available, this procedure can be applied immediately to the precise dissection and analysis of genomic DNA from any source and to any other research problem requiring efficient, highly specific cleavage of DNA at predetermined sites.     

PcrA-mediated disruption of RecA nucleoprotein filaments--essential role of the ATPase activity of RecA

The essential DNA helicase, PcrA, regulates recombination by displacing the recombinase RecA from the DNA. The nucleotide-bound state of RecA determines the stability of its nucleoprotein filaments. Using single-molecule fluorescence approaches, we demonstrate that RecA displacement by a translocating PcrA requires the ATPase activity of the recombinase. We also show that in a 'head-on collision' between a polymerizing RecA filament and a translocating PcrA, the RecA K72R ATPase mutant, but not wild-type RecA, arrests helicase translocation. Our findings demonstrate that translocation of PcrA is not sufficient to displace RecA from the DNA and assigns an essential role for the ATPase activity of RecA in helicase-mediated disruption of its filaments.     

The differential extension in dsDNA bound to Rad51 filaments may play important roles in homology recognition and strand exchange

RecA and Rad51 proteins play an important role in DNA repair and homologous recombination. For RecA, X-ray structure information and single molecule force experiments have indicated that the differential extension between the complementary strand and its Watson–Crick pairing partners promotes the rapid unbinding of non-homologous dsDNA and drives strand exchange forward for homologous dsDNA. In this work we find that both effects are also present in Rad51 protein. In particular, pulling on the opposite termini (3′ and 5′) of one of the two DNA strands in a dsDNA molecule allows dsDNA to extend along non-homologous Rad51-ssDNA filaments and remain stably bound in the extended state, but pulling on the 3′5′ ends of the complementary strand reduces the strand-exchange rate for homologous filaments. Thus, the results suggest that differential extension is also present in dsDNA bound to Rad51. The differential extension promotes rapid recognition by driving the swift unbinding of dsDNA from non-homologous Rad51-ssDNA filaments, while at the same time, reducing base pair tension due to the transfer of the Watson–Crick pairing of the complementary strand bases from the highly extended outgoing strand to the slightly less extended incoming strand, which drives strand exchange forward.     

A RecA filament capping mechanism for RecX protein

The RecX protein is a potent inhibitor of RecA protein activities. RecX functions by specifically blocking the extension of RecA filaments. In vitro, this leads to a net disassembly of RecA protein from circular single-stranded DNA. Based on multiple observations, we propose that RecX has a RecA filament capping activity. This activity has predictable effects on the formation and disassembly of RecA filaments. In vivo, the RecX protein may limit the length of RecA filaments formed during recombinational DNA repair and other activities. RecX protein interacts directly with RecA protein, but appears to interact in a functionally significant manner only with RecA filaments bound to DNA.     

Inhibition of RecA protein function by the RdgC protein from Escherichia coli

The Escherichia coli RdgC protein is a potential negative regulator of RecA function. RdgC inhibits RecA protein-promoted DNA strand exchange, ATPase activity, and RecA-dependent LexA cleavage. The primary mechanism of RdgC inhibition appears to involve a simple competition for DNA binding sites, especially on duplex DNA. The capacity of RecA to compete with RdgC is improved by the DinI protein. RdgC protein can inhibit DNA strand exchange catalyzed by RecA nucleoprotein filaments formed on single-stranded DNA by binding to the homologous duplex DNA and thereby blocking access to that DNA by the RecA nucleoprotein filaments. RdgC protein binds to single-stranded and double-stranded DNA, and the protein can be visualized on DNA using electron microscopy. RdgC protein exists in solution as a mixture of oligomeric states in equilibrium, most likely as monomers, dimers, and tetramers. This concentration-dependent change of state appears to affect its mode of binding to DNA and its capacity to inhibit RecA. The various species differ in their capacity to inhibit RecA function.     

Disassembly of Escherichia coli RecA E38K/��C17 Nucleoprotein Filaments Is Required to Complete DNA Strand Exchange

Disassembly of RecA protein subunits from a RecA filament has long been known to occur during DNA strand exchange, although its importance to this process has been controversial. An Escherichia coli RecA E38K/ΔC17 double mutant protein displays a unique and pH-dependent mutational separation of DNA pairing and extended DNA strand exchange. Single strand DNA-dependent ATP hydrolysis is catalyzed by this mutant protein nearly normally from pH 6 to 8.5. It will also form filaments on DNA and promote DNA pairing. However, below pH 7.3, ATP hydrolysis is completely uncoupled from extended DNA strand exchange. The products of extended DNA strand exchange do not form. At the lower pH values, disassembly of RecA E38K/ΔC17 filaments is strongly suppressed, even when homologous DNAs are paired and available for extended DNA strand exchange. Disassembly of RecA E38K/ΔC17 filaments improves at pH 8.5, whereas complete DNA strand exchange is also restored. Under these sets of conditions, a tight correlation between filament disassembly and completion of DNA strand exchange is observed. This correlation provides evidence that RecA filament disassembly plays a major role in, and may be required for, DNA strand exchange. A requirement for RecA filament disassembly in DNA strand exchange has a variety of ramifications for the current models linking ATP hydrolysis to DNA strand exchange.     

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.     

Structure of helical RecA-DNA complexes. Complexes formed in the presence of ATP-gamma-S or ATP

Electron micrographs of RecA-DNA filaments, formed under several different conditions, have been analyzed and the filament images reconstructed in three dimensions. In the presence of ATP and a non-hydrolyzable ATP analog. ATP-gamma-S, the RecA protein forms with DNA a right-handed helical complex with a pitch of approximately 95 A. The most detailed view of the filament was obtained from analysis of RecA filaments on double-stranded DNA in the presence of ATP-gamma-S. There are approximately six subunits of RecA per turn of the helix, but both this number and the pitch are variable. From the examination of single filaments and filament-filament interactions, a picture of an extremely flexible protein structure emerges. The subunits of RecA protein are seen to be arranged in such a manner that the bound DNA must be partially exposed and able to come into contact with external DNA molecules. The RecA structure determined in the presence of ATP-gamma-S appears to be the same as the "pre-synaptic" state that occurs with ATP, in which there is recognition and pairing between homologous DNA molecules.     

Multiplex PCR: use of heat-stable Thermus thermophilus RecA protein to minimize non-specific PCR products

In this paper we report that the inclusion of heat-resistant RecA protein from a thermophilic bacteria, Thermus thermophilus, and its cofactor (ATP) in PCR effectively eliminates non-specific PCR products. The effect of RecA protein, which catalyzes pairing between homologous DNA molecules with great fidelity in genetic recombination, is due to its promotion of precise priming in PCR (i.e. priming at sites where the primer sequence is completely complementary to that of the target sequence). In addition, the RecA protein substantially reduces the primer concentration required for PCR. These experimental results have led to the realization of multiplex PCR, which involves PCR for multiple sites in the same reaction mixture. We were able to successfully perform multiplex PCR with over a dozen reactions without affecting the amplification pattern of the PCR products.     

RecA dimers serve as a functional unit for assembly of active nucleoprotein filaments

All RecA-like recombinase enzymes catalyze DNA strand exchange as elongated filaments on DNA. Despite numerous biochemical and structural studies of RecA and the related Rad51 and RadA proteins, the unit oligomer(s) responsible for nucleoprotein filament assembly and coordinated filament activity remains undefined. We have created a RecA fused dimer protein and show that it maintains in vivo DNA repair and LexA co-protease activities, as well as in vitro ATPase and DNA strand exchange activities. Our results support the idea that dimeric RecA is an important functional unit both for assembly of nucleoprotein filaments and for their coordinated activity during the catalysis of homologous recombination.     

The human Rad51 protein: polarity of strand transfer and stimulation by hRP-A.

The human Rad51 protein is homologous to the RecA protein and catalyses homologous pairing and strand transfer reactions in vitro. Using single-stranded circular and homologous linear duplex DNA, we show that hRad51 forms stable joint molecules by transfer of the 5' end of the complementary strand of the linear duplex to the ssDNA. The polarity of strand transfer is therefore 3' to 5', defined relative to the ssDNA on which hRad51 initiates filament formation. This polarity is opposite to that observed with RecA. Homologous pairing and strand transfer require stoichiometric amounts of hRad51, corresponding to one hRad51 monomer per three nucleotides of ssDNA. Joint molecules are not observed when the protein is present in limiting or excessive amounts. The human ssDNA binding-protein, hRP-A, stimulates hRad51-mediated reactions. Its effect is consistent with a role in the removal of secondary structures from ssDNA, thereby facilitating the formation of continuous Rad51 filaments.     

Rad52 promotes postinvasion steps of meiotic double-strand-break repair

During DNA double-strand-break (DSB) repair by recombination, the broken chromosome uses a homologous chromosome as a repair template. Early steps of recombination are well characterized: DSB ends assemble filaments of RecA-family proteins that catalyze homologous pairing and strand-invasion reactions. By contrast, the postinvasion steps of recombination are poorly characterized. Rad52 plays an essential role during early steps of recombination by mediating assembly of a RecA homolog, Rad51, into nucleoprotein filaments. The meiosis-specific RecA-homolog Dmc1 does not show this dependence, however. By exploiting the Rad52 independence of Dmc1, we reveal that Rad52 promotes postinvasion steps of both crossover and noncrossover pathways of meiotic recombination in Saccharomyces cerevisiae. This activity resides in the N-terminal region of Rad52, which can anneal complementary DNA strands, and is independent of its Rad51-assembly function. Our findings show that Rad52 functions in temporally and biochemically distinct reactions and suggest a general annealing mechanism for reuniting DSB ends during recombination.     

Structural analysis of RecA protein-DNA complexes by fluorescence-detected linear dichroism: absence of structural change of filament for pairing of complementary DNA strands

We have developed a simple measuring system for fluorescence-detected linear dichroism and applied it to the structural analysis of the RecA-DNA complex filaments, which are intermediates of the homologous recombination reaction. Taking advantage of the selectivity of fluorescence signals, we distinguished the linear dichroism signals of ethidium bromide and tryptophan residues in the RecA-DNA-ethidium bromide complex, whereas the conventional (absorption-detected) linear dichroism measurement provides only the sum of the signals because signals overlap each other and that of DNA. We further observed that the tryptophan residue at position 290 of RecA in the RecA-DNA-adenosine-5'-O-(3-thiotriphosphate) complex was oriented parallel to the long axis of the filament, in good agreement with the previous site-specific linear dichroism analysis, and that this orientation was not significantly modified by the pairing of the complementary DNA strand. These results suggest that the pairing reaction occurs without a large structural change of the RecA filament.     

The Rad51-dependent pairing of long DNA substrates is stabilized by replication protein A

Rad51 protein forms nucleoprotein filaments on single-stranded DNA (ssDNA) and then pairs that DNA with the complementary strand of incoming duplex DNA. In apparent contrast with published results, we demonstrate that Rad51 protein promotes an extensive pairing of long homologous DNAs in the absence of replication protein A. This pairing exists only within the Rad51 filament; it was previously undetected because it is lost upon deproteinization. We further demonstrate that RPA has a critical postsynaptic role in DNA strand exchange, stabilizing the DNA pairing initiated by Rad51 protein. Stabilization of the Rad51-generated DNA pairing intermediates can be can occur either by binding the displaced strand with RPA or by degrading the same DNA strand using exonuclease VII. The optimal conditions for Rad51-mediated DNA strand exchange used here minimize the secondary structure in single-stranded DNA, minimizing the established presynaptic role of RPA in facilitating Rad51 filament formation. We verify that RPA has little effect on Rad51 filament formation under these conditions, assigning the dramatic stimulation of strand exchange nevertheless afforded by RPA to its postsynaptic function of removing the displaced DNA strand from Rad51 filaments.     

ATP-mediated changes in cross-subunit interactions in the RecA protein

RecA protein undergoes ATP- and DNA-induced conformational changes that result in different helical parameters for free protein filaments versus RecA/ATP/DNA nucleoprotein filaments. Previous mutational studies of a particular region of the RecA oligomeric interface suggested that cross-subunit contacts made by residues K6 and R28 were more important for stabilization of free protein oligomers than nucleoprotein filaments [Eldin, S., et al. (2000) J. Mol. Biol. 299, 91-101]. Using mutant proteins with specifically engineered Cys substitutions, we show here that the efficiency of cross-subunit disulfide bond formation at certain positions in this region changes in the presence of ATP or ATP/DNA. Our results support the idea that specific cross-subunit interactions that occur within this region of the subunit interface are different in free RecA protein versus RecA/ATP/DNA nucleoprotein filaments.