Exchange of DNA base pairs that coincides with recognition of homology promoted by E. coli RecA protein

The unresolved mechanism by which a single strand of DNA recognizes homology in duplex DNA is central to understanding genetic recombination and repair of double-strand breaks. Using stopped-flow fluorescence we monitored strand exchange catalyzed by E. coli RecA protein, measuring simultaneously the rate of exchange of A:T base pairs and the rates of formation and dissociation of the three-stranded intermediates called synaptic complexes. The rate of exchange of A:T base pairs was indistinguishable from the rate of formation of synaptic complexes, whereas the rate of displacement of a single strand from complexes was five to ten times slower. This physical evidence shows that a subset of bases exchanges at a rate that is fast enough to account for recognition of homology. Together, several studies suggest that a mechanism governed by the dynamic structure of DNA and catalyzed by diverse enzymes underlies both recognition of homology and initiation of strand exchange.     

Heterology tolerance and recognition of mismatched base pairs by human Rad51 protein

Human Rad51 (hRad51) promoted homology recognition and subsequent strand exchange are the key steps in human homologous recombination mediated repair of DNA double-strand breaks. However, it is still not clear how hRad51 deals with sequence heterology between the two homologous chromosomes in eukaryotic cells, which would lead to mismatched base pairs after strand exchange. Excessive tolerance of sequence heterology may compromise the fidelity of repair of DNA double-strand breaks. In this study, fluorescence resonance energy transfer (FRET) was used to monitor the heterology tolerance of human Rad51 mediated strand exchange reactions, in real time, by introducing either G-T or I-C mismatched base pairs between the two homologous DNA strands. The strand exchange reactions were much more sensitive to G-T than to I-C base pairs. These results imply that the recognition of homology and the tolerance of heterology by hRad51 may depend on the local structural motif adopted by the base pairs participating in strand exchange. AnhRad51 mutant protein (hRad51K133R), deficient in ATP hydrolysis, showed greater heterology tolerance to both types of mismatch base pairing, suggesting that ATPase activity may be important for maintenance of high fidelity homologous recombination DNA repair.     

High fidelity of RecA-catalyzed recombination: a watchdog of genetic diversity

Homologous recombination plays a key role in generating genetic diversity, while maintaining protein functionality. The mechanisms by which RecA enables a single-stranded segment of DNA to recognize a homologous tract within a whole genome are poorly understood. The scale by which homology recognition takes place is of a few tens of base pairs, after which the quest for homology is over. To study the mechanism of homology recognition, RecA-promoted homologous recombination between short DNA oligomers with different degrees of heterology was studied in vitro, using fluorescence resonant energy transfer. RecA can detect single mismatches at the initial stages of recombination, and the efficiency of recombination is strongly dependent on the location and distribution of mismatches. Mismatches near the 5′ end of the incoming strand have a minute effect, whereas mismatches near the 3′ end hinder strand exchange dramatically. There is a characteristic DNA length above which the sensitivity to heterology decreases sharply. Experiments with competitor sequences with varying degrees of homology yield information about the process of homology search and synapse lifetime. The exquisite sensitivity to mismatches and the directionality in the exchange process support a mechanism for homology recognition that can be modeled as a kinetic proofreading cascade.     

Hallmarks of homology recognition by RecA-like recombinases are exhibited by the unrelated Escherichia coli RecT protein

Homologous recombination is a fundamental process for genome maintenance and evolution. Various proteins capable of performing homology recognition and pairing of DNA strands have been isolated from many organisms. The RecA family of proteins exhibits a number of biochemical properties that are considered hallmarks of homology recognition. Here, we investigated whether the unrelated Escherichia coli RecT protein, which mediates homologous pairing and strand exchange, also exhibits such properties. We found that, like RecA and known RecA homologs: (i) RecT promotes the co-aggregation of ssDNA with duplex DNA, which is known to facilitate homologous contacts; (ii) RecT binding to ssDNA mediates unstacking of the bases, a key step in homology recognition; (iii) RecT mediates the formation of a three-strand synaptic intermediate where pairing is facilitated by local helix destabilization, and the preferential switching of A:T base pairs mediates recognition of homology; and (iv) RecT-mediated pairing occurs from both 3'- and 5'-single-stranded ends. Taken together, our results show that RecT shares fundamental homology-recognition properties with the RecA homologs, and provide new insights on an underlying universal mechanism of homologous recognition.     

Mismatch recognition in chromosomal interactions and speciation

Homologous chromosomes interact during meiosis by means of proteins involved in recombination and in the recognition and repair of mismatched base pairs. Recombination proteins bring homologous chromosomes or chromosomal regions together by facilitating the search for DNA homology and by catalyzing strand exchange between homologous molecules or regions. Mismatch recognition and repair proteins act as editors of recombination and appear to disrupt those DNA associations that contain mismatched base pairs. Thus, it may be that, as chromosomes diverge in their primary sequence and become increasingly polymorphic, recombinational interactions leading to chromosome pairing and recombination tend to be inhibited. Decreasing homologous interactions within and between chromosomes will clearly contribute to maintaing the integrity of individual chromosomes and may utimately lead, as a result of sterile meioses, to the reproductive isolation of closely related species.     

RecA protein-facilitated DNA strand breaks. A mechanism for bypassing DNA structural barriers during strand exchange

RecA protein promotes an unexpectedly efficient DNA strand exchange between circular single-stranded DNA and duplex DNAs containing short (50-400-base pair) heterologous sequences at the 5' (initiating) end. The major mechanism by which this topological barrier is bypassed involves DNA strand breakage. Breakage is both strand and position specific, occurring almost exclusively in the displaced (+) strand of the duplex within a 15-base pair region of the heterology/homology junction. Breakage also requires recA protein, ATP hydrolysis, and homologous sequences 3' to the heterology. Although the location of the breaks and the observed requirements clearly indicate a major role for recA protein in this phenomenon, the molecular mechanism is not yet clear. The breakage may reflect a DNA structure and/or some form of structural stress within the DNA during recA protein-mediated DNA pairing which either exposes the DNA at this precise position to the action of a contaminating nuclease or induces a direct mechanical break. We also find that when heterology is located at the 3' end of the linear duplex, strand exchange is halted (without DNA breakage) about 500 base pairs from the homology/heterology junction.     

Integrating multi-scale data on homologous recombination into a new recognition mechanism based on simulations of the RecA-ssDNA/dsDNA structure

RecA protein is the prototypical recombinase. Members of the recombinase family can accurately repair double strand breaks in DNA. They also provide crucial links between pairs of sister chromatids in eukaryotic meiosis. A very broad outline of how these proteins align homologous sequences and promote DNA strand exchange has long been known, as are the crystal structures of the RecA-DNA pre- and postsynaptic complexes; however, little is known about the homology searching conformations and the details of how DNA in bacterial genomes is rapidly searched until homologous alignment is achieved. By integrating a physical model of recognition to new modeling work based on docking exploration and molecular dynamics simulation, we present a detailed structure/function model of homology recognition that reconciles extremely quick searching with the efficient and stringent formation of stable strand exchange products and which is consistent with a vast body of previously unexplained experimental results.     

From strand exchange to branch migration; bypassing of non-homologous sequences by human Rad51 and Rad54

Rad51 and Rad54 play crucial roles during homologous recombination. The biochemical activities of human Rad51 (hRad51) and human Rad54 (hRad54) and their interactions with each other are well documented. However, it is not known how these two proteins work together to bypass heterologous sequences; i.e. mismatched base pairs, during homologous recombination. In this study, we used a fluorescence resonance energy transfer assay to monitor homologous recombination processes in real time so that the interactions between hRad54 and hRad51 during DNA strand exchange and branch migration, which are two core steps of homologous recombination, could be characterized. Our results indicate that hRad54 can facilitate hRad51-promoted strand exchange through various degrees of mismatching. We propose that the main roles of hRad51 in homologous recombination is to initiate the homology recognition and strand-exchange steps and those of hRad54 are to promote efficient branch migration, bypass potential mismatches and facilitate long-range strand exchanges through branch migration of Holliday junctions.     

The poor homology stringency in the heteroduplex allows strand exchange to incorporate desirable mismatches without sacrificing recognition in vivo

RecA family proteins are responsible for homology search and strand exchange. In bacteria, homology search begins after RecA binds an initiating single-stranded DNA (ssDNA) in the primary DNA-binding site, forming the presynaptic filament. Once the filament is formed, it interrogates double-stranded DNA (dsDNA). During the interrogation, bases in the dsDNA attempt to form Watson–Crick bonds with the corresponding bases in the initiating strand. Mismatch dependent instability in the base pairing in the heteroduplex strand exchange product could provide stringent recognition; however, we present experimental and theoretical results suggesting that the heteroduplex stability is insensitive to mismatches. We also present data suggesting that an initial homology test of 8 contiguous bases rejects most interactions containing more than 1/8 mismatches without forming a detectable 20 bp product. We propose that, in vivo, the sparsity of accidental sequence matches allows an initial 8 bp test to rapidly reject almost all non-homologous sequences. We speculate that once the initial test is passed, the mismatch insensitive binding in the heteroduplex allows short mismatched regions to be incorporated in otherwise homologous strand exchange products even though sequences with less homology are eventually rejected.     

Patterns of lambda Int recognition in the regions of strand exchange

Int protein has two classes of binding sites within the phage att site: the arm-type recognition sequences are found in three specific sites that are distant from the region of strand exchange; the junction-type recognition sequences occur as inverted pairs around the crossover region in both attP and attB. During recombination between attP and attB each of the four DNA strands is cut at a homologous position within each of the junction-type Int binding sites. In all four junction-type sites Int protein interacts primarily with the same face of the DNA helix, as determined by those purine nitrogens that are protected against methylation by dimethylsulfate. Efficient secondary attachment sites for lambda contain sequences with partial homology to the junction-type binding sites. In addition, the sequence between, but not part of, the two junction-type sites (the overlap region) is strongly conserved in secondary att sites. Thus, in the vicinity of strand exchange, attP and a recombining partner, such as attB, are very similar; each comprises two junction-type Int recognition sites and an overlap (crossover) region.     

Homology-dependent changes in adenosine 5'-triphosphate hydrolysis during recA protein promoted DNA strand exchange: evidence for long paranemic complexes

As a first step in DNA strand exchange, recA protein forms a filamentous complex on single-stranded DNA (ssDNA), which contains stoichiometric (one recA monomer per four nucleotides) amounts of recA protein. recA protein monomers within this complex hydrolyze ATP with a turnover number of 25 min-1. Upon introduction of linear homologous duplex DNA to initiate strand exchange, this rate of ATP hydrolysis drops by 33%. The decrease in rate is complete in less than 2 min, and the rate of ATP hydrolysis then remains constant during and subsequent to the strand exchange reaction. This drop is completely dependent upon homology in the duplex DNA. In addition, the magnitude of the drop is linearly dependent upon the length of the homologous region in the linear duplex DNA. Linear DNA substrates in which pairing is topologically restricted to a paranemic joint also follow this relationship. Taken together, these properties imply that all of the available homology in the incoming duplex DNA is detected very early in the DNA strand exchange reaction, with the linear duplex DNA paired paranemically with the homologous ssDNA in the complex throughout its length. The results indicate that paranemic joints can extend over thousands of base pairs. We note elsewhere [Pugh, B. F., & Cox, M. M. (1987b) J. Biol. Chem. 262, 1337-1343] that this duplex acquires resistance to digestion by DNase with a much slower time course (30 min), which parallels the progress of strand exchange. Together these results imply that the duplex DNA is paired with the ssDNA but remains outside the nucleoprotein filament. Finally, the results also support the notion that ATP hydrolysis occurs throughout the recA nucleoprotein filament.     

A chimeric Rec-A protein that implicates non-Watson-Crick interactions in homologous pairing.

The helical filament formed by RecA protein on single-stranded DNA plays an important role in homologous recombination and pairs with a complementary single strand or homologous duplex DNA. The RecA nucleoprotein filament also recognizes an identical single strand. The chimeric protein, RecAc38, forms a nucleoprotein filament that recognizes a complementary strand but is defective in recognition of duplex DNA, and is associated with phenotypic defects in repair and recombination. As described here, RecAc38 nucleoprotein filament is also defective in recognition of an identical strand, either when the filament has within it a single strand or duplex DNA. A model that postulates three DNA binding sites rationalizes these observations and suggests that the third binding site mediates non-Watson-Crick interactions that are instrumental in recognition of homology in duplex DNA.     

Reversible dimer formation and stability of the anti-tumour single-chain Fv antibody MFE-23 by neutron scattering,analytical ultracentrifugation,and NMR and FT-IR spectroscopy

Cre recombinase uses two pairs of sequential cleavage and religation reactions to exchange homologous DNA strands between 34 base-pair (bp) LoxP recognition sequences. In the oligomeric recombination complex, a switch between "cleaving" and "non-cleaving" subunit conformations regulates the number, order, and regio-specificity of the strand exchanges. However, the particular sequence of events has been in question. From analysis of strand composition of the Holliday junction (HJ) intermediate, we determined that Cre initiates recombination of LoxP by cleaving the upper strand on the left arm. Cre preferred to react with the left arm of a LoxP suicide substrate, but at a similar rate to the right arm, indicating that the first strand to be exchanged is selected prior to cleavage. We propose that during complex assembly the cleaving subunit preferentially associates with the LoxP left arm, directing the first strand exchange to that side. In addition, this biased assembly would enforce productive orientation of LoxP sites in the recombination synapses. A novel Cre-HJ complex structure in which LoxP was oriented with the left arm bound by the cleaving Cre subunit suggested a physical basis for the strand exchange order. Lys86 and Lys201 interact with the left arm scissile adenine base differently than in structures that have a scissile guanine. These interactions are associated with positioning the 198-208 loop, a structural component of the conformational switch, in a configuration that is specific to the cleaving conformation. Our results suggest that strand exchange order and site alignment are regulated by an "induced fit" mechanism in which the cleaving conformation is selectively stabilized through protein-DNA interactions with the scissile base on the strand that is cleaved first.     

Dynamic opening of DNA during the enzymatic search for a damaged base

Uracil DNA glycosylase (UDG) removes uracil from U.A or U.G base pairs in genomic DNA by extruding the aberrant uracil from the DNA base stack. A question in enzymatic DNA repair is whether UDG and related glycosylases also use an extrahelical recognition mechanism to inspect the integrity of undamaged base pairs. Using NMR imino proton exchange measurements we find that UDG substantially increases the equilibrium constant for opening of T-A base pairs by almost two orders of magnitude relative to free B-DNA. This increase is brought about by enzymatic stabilization of an open state of the base pair without increasing the rate constant for spontaneous base pair opening. These findings indicate a passive search mechanism in which UDG uses the spontaneous opening dynamics of DNA to inspect normal base pairs in a rapid genome-wide search for uracil in DNA.     

Complementary strand relocation may play vital roles in RecA-based homology recognition

RecA-family proteins mediate homologous recombination and recombinational DNA repair through homology search and strand exchange. Initially, the protein forms a filament with the incoming single-stranded DNA (ssDNA) bound in site I. The RecA–ssDNA filament then binds double-stranded DNA (dsDNA) in site II. Non-homologous dsDNA rapidly unbinds, whereas homologous dsDNA undergoes strand exchange yielding heteroduplex dsDNA in site I and the leftover outgoing strand in site II. We show that applying force to the ends of the complementary strand significantly retards strand exchange, whereas applying the same force to the outgoing strand does not. We also show that crystallographically determined binding site locations require an intermediate structure in addition to the initial and final structures. Furthermore, we demonstrate that the characteristic dsDNA extension rates due to strand exchange and free RecA binding are the same, suggesting that relocation of the complementary strand from its position in the intermediate structure to its position in the final structure limits both rates. Finally, we propose that homology recognition is governed by transitions to and from the intermediate structure, where the transitions depend on differential extension in the dsDNA. This differential extension drives strand exchange forward for homologs and increases the free energy penalty for strand exchange of non-homologs.     

DNA strand exchange catalyzed by proteins from vaccinia virus-infected cells.

Vaccinia virus infection induces expression of a protein which can catalyze joint molecule formation between a single-stranded circular DNA and a homologous linear duplex. The kinetics of appearance of the enzyme parallels that of vaccinia virus DNA polymerase and suggests it is an early viral gene product. Extracts were prepared from vaccinia virus-infected HeLa cells, and the strand exchange assay was used to follow purification of this activity through five chromatographic steps. The most highly purified fraction contained three major polypeptides of 110 +/- 10, 52 +/- 5, and 32 +/- 3 kDa. The purified protein requires Mg2+ for activity, and this requirement cannot be satisfied by Mn2+ or Ca2+. One end of the linear duplex substrate must share homology with the single-stranded circle, although this homology requirement is not very high, as 10% base substitutions had no effect on the overall efficiency of pairing. As with many other eukaryotic strand exchange proteins, there was no requirement for ATP, and ATP analogs were not inhibitors. Electron microscopy was used to show that the joint molecules formed in these reactions were composed of a partially duplex circle of DNA bearing a displaced single-strand and a duplex linear tail. The recovery of these structures shows that the enzyme catalyzes true strand exchange. There is also a unique polarity to the strand exchange reaction. The enzyme pairs the 3' end of the duplex minus strand with the plus-stranded homolog, thus extending hybrid DNA in a 3'-to-5' direction with respect to the minus strand. Which viral gene (if any) encodes the enzyme is not yet known, but analysis of temperature-sensitive mutants shows that activity does not require the D5R gene product. Curiously, v-SEP appears to copurify with vaccinia virus DNA polymerase, although the activities can be partially resolved on phosphocellulose columns.     

DNase protection by recA protein during strand exchange. Asymmetric protection of the Holliday structure

When recA protein pairs linear duplex DNA with a homologous duplex molecule that has a single-stranded tail, it produces a recombination intermediate called the Holliday structure and causes reciprocal or symmetric strand exchange, whereas the pairing of a linear duplex molecule with fully single-stranded DNA leads to an asymmetric exchange. To study the location of recA protein on DNA molecules undergoing symmetric exchange, we labeled individually each end of the four strands involved and looked for protection against DNase I or restriction endonucleases. As expected, because of its preferred binding to single-stranded DNA, recA protein protected the single-stranded tails of either substrates, or products. In addition however, strong protection extended into the newly formed heteroduplex DNA along the strand to which recA protein was initially bound. Experiments with uniformly labeled DNA showed a corresponding homology-dependent asymmetry in the protection of the tailed substrate versus its fully duplex partner. Restriction experiments showed that protection extended 50-75 base pairs beyond the point where strand exchange was blocked by a long region of heterology. When compared with earlier observations (Chow, S. A., Honigberg, S. M., Bainton, R. J., and Radding, C. M. (1986) J. Biol. Chem. 261, 6961-6971), the present experiments reveal a pattern of association of recA protein with DNA that suggests a common mechanism of asymmetric and symmetric strand exchange.     

Features of two hepatitis B virus (HBV) DNA integrations suggest mechanisms of HBV integration.

Two integrated hepatitis B virus (HBV) DNA molecules were cloned from two primary hepatocellular carcinomas each containing only a single integration. One integration (C3) contained a single linear segment of HBV DNA, and the other integration (C4) contained a large inverted duplication of viral DNA at the site of a chromosome translocation (O. Hino, T.B. Shows, and C.E. Rogler, Proc. Natl. Acad. Sci. USA 83:8338-8342, 1986). Sequence analysis of the virus-cell junctions of C3 placed the left virus-cell junction at nucleotide 1824, which is at the 5' end of the directly repeated DR1 sequence and is 6 base pairs from the 3' end of the long (L) negative strand. The right virus-cell junction was at nucleotide 1762 in a region of viral DNA (within the cohesive overlap) which shared 5-base-pair homology with cellular DNA. Sequence analysis of the normal cellular DNA across the integration site showed that 11 base pairs of cellular DNA were deleted at the site of integration. On the basis of this analysis, we suggest a mechanism for integration of the viral DNA molecule which involves strand invasion of the 3' end of the L negative strand of an open circular or linear HBV DNA molecule (at the DR1 sequence) and base pairing of the opposite end of the molecule with cellular DNA, accompanied by the deletion of 11 base pairs of cellular DNA during the double recombination event. Sequencing across the inverted duplication of HBV DNA in clone C4 located one side of the inversion at nucleotide 1820, which is 2 base pairs from the 3' end of the L negative strand. Both this sequence and the left virus-cell junction of C3 are within the 9-nucleotide terminally redundant region of the HBV L negative strand DNA. We suggest that the terminal redundancy is a preferred topoisomerase I nicking region because of both its base sequence and forked structure. Such nicking would lead to integration and rearrangement of HBV molecules within the terminal redundancy, as we have observed in both our clones.     

Patterns of nuclease protection during strand exchange. recA protein forms heteroduplex DNA by binding to strands of the same polarity

recA protein, in the presence of ATP, polymerizes on single-stranded DNA (plus strand) to form a presynaptic nucleoprotein filament that pairs with linear duplex DNA and actively displaces the plus strand from the recipient molecule in a polarized fashion to form a new heteroduplex molecule. The interaction between recA protein and DNA during strand exchange was studied by labeling different strands and probing the intermediate with pancreatic deoxyribonuclease I (DNase I) or restriction endonuclease. The incoming single strand was resistant to DNase I in the original nucleoprotein filament and remained resistant even after extensive strand exchange had occurred. Both strands of the parental duplex molecule were sensitive to DNase I in the absence of joint molecule formation; but as strand exchange progressed following homologous pairing, increasing stretches of the parental plus strand became resistant, whereas the complementary parental minus strand remained sensitive to DNase I throughout the reaction. Except for a region of 50-100 base pairs at the end of the newly formed heteroduplex DNA where strand exchange was initiated, the rest of the heteroduplex region was resistant to cleavage by restriction endonucleases. The data suggest that recA protein promotes strand exchange by binding both the incoming and outgoing strands of the same polarity, whereas the complementary strand, which must switch pairing partners, is unhindered by direct contact with the protein.     

RecA homology search is promoted by mechanical stress along the scanned duplex DNA

A RecA-single-stranded DNA (RecA-ssDNA) filament searches a genome for sequence homology by rapidly binding and unbinding double-stranded DNA (dsDNA) until homology is found. We demonstrate that pulling on the opposite termini (3' and 5') of one of the two DNA strands in a dsDNA molecule stabilizes the normally unstable binding of that dsDNA to non-homologous RecA-ssDNA filaments, whereas pulling on the two 3', the two 5', or all four termini does not. We propose that the 'outgoing' strand in the dsDNA is extended by strong DNA-protein contacts, whereas the 'complementary' strand is extended by the tension on the base pairs that connect the 'complementary' strand to the 'outgoing' strand. The stress resulting from different levels of tension on its constitutive strands causes rapid dsDNA unbinding unless sufficient homology is present.