The role of single-stranded DNA in Flp-mediated strand exchange

The Flp recognition target site contains two inverted 13-base pair (bp) Flp binding sequences that surround an 8-bp core region. Flp recombinase has been shown to carry out strand ligation independently of its ability to execute strand cleavage. Using a synthetic activated DNA substrate bearing a 3'-phosphotyrosine group, we have developed an assay to measure strand exchange by Flp proteins. We have shown that wild-type Flp protein was able to catalyze strand exchange using DNA substrates containing 8-bp duplex core sequences. Mutant Flp proteins that are defective in either DNA bending or DNA cleavage were also impaired in their abilities to carry out strand exchange. The inability of these mutant proteins to execute strand exchange could be overcome by providing a DNA substrate containing a single-stranded core sequence. This single-stranded core sequence could be as small as 3 nucleotides. Full activity of mutant Flp proteins in strand exchange was observed when both partner DNAs contained an 8-nucleotide single-stranded core region. Using suicide substrates, we showed that single-stranded DNA is also important for strand exchange reactions where Flp-mediated strand cleavage is required. These results suggest that the ability of Flp to induce DNA bending and strand cleavage may be crucial for strand exchange. We propose that both DNA bending and strand cleavage may be required to separate the strands of the core region and that single-stranded DNA in the core region might be an intermediate in Flp-mediated DNA recombination.     

The Flp recombinase cleaves Holliday junctions in trans

The Flp site-specific recombinase is encoded by the 2 micrometers plasmid Saccharomyces cerevisiae and is a member of the integrase family of recombinases. Like all members of the integrase family studied, Flp mediates recombination in two steps. First, a pair of strand exchanges creates a Holliday-like intermediate; second, this intermediate is resolved to recombinant products by a second pair of strand exchanges. Evidence derived from experiments using linear substrates indicates that Flp's active site is composed of two Flp protomers. One binds to the Flp recognition target site (FRT site) and activates the scissile phosphodiester bond for cleavage. Another molecule of Flp bound elsewhere in the synaptic complex (in trans) donates the nucleophilic tyrosine that executes cleavage and thereby becomes covalently attached to the 3' phosphoryl group at the cleavage site. It has previously been shown that Flp efficiently resolves synthetic, Holliday-like (chi) structures to linear products. In this paper, we examined whether resolution of chi structures by Flp also occurs via the trans cleavage mechanism. We used in vitro complementation studies of mutant Flp proteins as well as nicked chi structures to show that Flp resolves chi structures by trans cleavage. We propose a model for Flp-mediated recombination that incorporates trans cleavage at both the initial and resolution steps of strand exchange.     

A general model for site-specific recombination by the integrase family recombinases

We present here a general model for integrase family site-specific recombination using the geometric relationships of the cleavable phosphodiester bonds and the disposition of the recombinase monomers (defined by their binding planes) with respect to them. The 'oscillation model' is based largely on the conformations of the recombinase-bound DNA duplexes and their dynamics within Holliday junctions. The duplex substrate or the Holliday junction intermediate is capable of 'oscillating' between two cleavage-competent asymmetric states with respect to corres-ponding chemically inert 'equilibrium positions'. The model accommodates several features of the Flp system and predicts two modes of DNA cleavage during a normal recombination event. It is equally applicable to other systems that mediate recombination across 6, 7 or 8 bp long strand exchange regions (or spacers). The model is consistent with approximately 0-1, 1-2 and 2-3 bp of branch migration during recombination reactions involving 6, 7 and 8 bp spacers, respectively.     

Flp ribonuclease activities. Mechanistic similarities and contrasts to site-specific DNA recombination

The ribonuclease active site harbored by the Flp site-specific recombinase can act on two neighboring phosphodiester bonds to yield mechanistically distinct chain breakage reactions. One of the RNase reactions apparently proceeds via a covalent enzyme intermediate and targets the phosphodiester position involved in DNA recombination (Flp RNase I activity). The second activity (Flp RNase II) targets the phosphodiester immediately to the 3' side but appears not to involve an enzyme-linked intermediate. Flp RNase I is absolutely dependent upon Tyr-343 of Flp and is competitive with respect to the normal strand joining reaction. It can utilize the 2'-hydroxyl group from any one of the four ribonucleotides with comparable efficiencies in the cleavage reaction. On the other hand, the RNase II reaction mediated by Flp(Y343F) is specific for U and cannot utilize the 2'-hydroxyl group from ribo-A, -G, or -C under standard reaction conditions. The RNase II activity is also sensitive to the 3'-neighboring base. Although dT is functional, the activity is stimulated by U or U-2'-OMe. The Flp RNase II reaction effectively competes with the normal strand cleavage reaction mediated by Tyr-343, even though their phosphodiester targets are not the same.     

Sensing homology at the strand-swapping step in lambda excisive recombination

lambda Site-specific recombination requires a short stretch of sequence homology that might be sensed during strand swapping, during ligation and/or during isomerization of the obligate Holliday junction intermediate. Here, we use half-att site suicide substrates to study single and double top-strand-transfers, isolated from the subsequent steps of the reaction. The double-strand-transfer is analogous to a top-strand exchange and consists of one normal top-strand and one "contrary" bottom-strand to top-strand ligation between the half-att site substrate and its full-site partner. The resulting covalent three-way DNA junctions are poor substrates for resolution in the forward or reverse direction. We show that both the rate and the efficiency of Y-junction formation are homology dependent. Pairing of three nucleotides (either in the forward or in the contrary alignment) provides maximal stability to strand swapping. Complementary base-pairing next to one top-strand site (with or without ligation) stimulates strand-transfer at the other mismatched site. The data suggest that homology can be sensed at the strand-swapping step before ligation. However, homology also stimulates ligation and stabilizes the products, as is evident from the different rates of closed Y-junction formation in the presence or absence of homology. Furthermore, under recombination conditions, single top-strand-transfers are subject to reversal even in the presence of sequence homology; stability depends on a double-strand-transfer, i.e. dissociation of covalent Int.     

The RAG-HMG1 complex enforces the 12/23 rule of V(D)J recombination specifically at the double-hairpin formation step

A central unanswered question concerning the initial phases of V(D)J recombination has been at which step the 12/23 rule applies. This rule, which governs which variable (V), diversity (D), and joining (J) segments are able to pair during recombination, could operate at the level of signal sequence synapsis after RAG-HMG1 complex binding, signal nicking, or signal hairpin formation. It has also been unclear whether additional proteins are required to achieve adherence to the 12/23 rule. We developed a novel system for the detailed biochemical analysis of the 12/23 rule by using an oligonucleotide-based substrate that can include two signals. Under physiologic conditions, we found that the complex of RAG1, RAG2, and HMG1 can successfully recapitulate the 12/23 rule with the same specificity as that seen intracellularly and in crude extracts. The cleavage complex can bind and nick 12x12 and 23x23 substrates as well as 12x23 substrates. However, hairpin formation occurs at both of the signals only on 12x23 substrates. Moreover, under physiologic conditions, the presence of a partner 23-bp spacer suppresses single-site hairpin formation at a 12-bp spacer and vice versa. Hence, this study illustrates that synapsis suppresses single-site reactions, thereby explaining the high physiologic ratio of paired versus unpaired V(D)J recombination events in lymphoid cells.     

Functional analysis of coordinated cleavage in V(D)J recombination

V(D)J recombination in vivo requires a pair of signals with distinct spacer elements of 12 and 23 bp that separate conserved heptamer and nonamer motifs. Cleavage in vitro by the RAG1 and RAG2 proteins can occur at individual signals when the reaction buffer contains Mn2+, but cleavage is restricted to substrates containing two signals when Mg2+ is the divalent cation. By using a novel V(D)J cleavage substrate, we show that while the RAG proteins alone establish a moderate preference for a 12/23 pair versus a 12/12 pair, a much stricter dependence of cleavage on the 12/23 signal pair is produced by the inclusion of HMG1 and competitor double-stranded DNA. The competitor DNA serves to inhibit the cleavage of substrates carrying a 12/12 or 23/23 pair, as well as the cutting at individual signals in 12/23 substrates. We show that a 23/33 pair is more efficiently recombined than a 12/33 pair, suggesting that the 12/23 rule can be generalized to a requirement for spacers that differ from each other by a single helical turn. Furthermore, we suggest that a fixed spatial orientation of signals is required for cleavage. In general, the same signal variants that can be cleaved singly can function under conditions in which a signal pair is required. However, a chemically modified substrate with one noncleavable signal enables us to show that formation of a functional cleavage complex is mechanistically separable from the cleavage reaction itself and that although cleavage requires a pair of signals, cutting does not have to occur simultaneously at both. The implications of these results are discussed with respect to the mechanism of V(D)J recombination and the generation of chromosomal translocations.     

Topological analysis of the role of homology in Flp-mediated recombination

Recombination by the Flp recombinase of Saccharomyces cerevisiae is known to be inhibited by heterology of the overlap regions of the two recombining DNA targets (FRT sites). We have used topological analysis to show that Flp can promote two rounds of intramolecular recombination between heterologous FRT sites contained within the same supercoiled plasmid. The products are in parental nonrecombinant configuration. Thus, heterology may appear to "block" recombination by rendering the heteroduplex recombinant products unstable, thus favoring a second round of recombination to homoduplex (but parental) products. Hence, homology in the core region is not a requirement for the recombination reaction by Flp but for the formation of recombinant products.     

Asymmetry in Flp-mediated cleavage

Flp is a member of the integrase family of site-specific recombinases. Members of the integrase family mediate DNA strand cleavage via a transesterification reaction involving an active site tyrosine residue. The first step of the reaction results in covalent linkage of the protein to the 3'-phosphoryl DNA terminus, leaving a 5'-hydroxyl group at the site of the nick. We have used Flp recognition target (FRT) sites containing a 5'-bridging phosphorothioate linkage at the site of Flp cleavage to accumulate intermediates in which Flp is covalently bound at a cleavage site. We have probed these intermediates with dimethylsulfate using methylation protection and find that Flp-mediated cleavage is associated with protection of two adenine residues that are opposite the sites of cleavage and covalent attachment by Flp. Methylation interference studies showed that cleavage and covalent attachment are also accompanied by differences in the contacts of Flp with each of the two cleavage sites and with the surrounding symmetry elements. Therefore, we provide evidence that Flp-mediated cleavage and covalent attachment result in changes to the conformation of the Flp-FRT complex. These changes may be required for Flp-mediated strand exchange activity.     

Three mechanistic steps detected by FRET after presynaptic filament formation in homologous recombination. ATP hydrolysis required for release of oligonucleotide heteroduplex product from RecA

The Escherichia coli RecA protein promotes DNA strand exchange in homologous recombination and recombinational DNA repair. Stopped-flow kinetics and fluorescence resonance energy transfer (FRET) were used to study RecA-mediated strand exchange between a 30-bp duplex DNA and a homologous single-stranded 50mer. In our standard assay, one end of the dsDNA helix was labeled at apposing 5' and 3' ends with hexachlorofluorescein and fluorescein, respectively. Strand exchange was monitored by the increase in fluorescence emission resulting upon displacement of the fluorescein-labeled strand from the initial duplex. The potential advantages of FRET in study of strand exchange are that it noninvasively measures real-time kinetics in the previously inaccessible millisecond time regime and offers great sensitivity. The oligonucleotide substrates model short-range mechanistic effects that might occur within a localized region of the ternary complex formed between RecA and long DNA molecules during strand exchange. Reactions in the presence of ATP with 0.1 microM duplex and 0.1-1.0 microM ss50mer showed triphasic kinetics in 600 s time courses, implying the existence of three mechanistic steps subsequent to presynaptic filament formation. The observed rate constants for the intermediate phase were independent of the concentration of ss50mer and most likely characterize a unimolecular isomerization of the ternary complex. The observed rate constants for the first and third phases decreased with increasing ss50mer concentration. Kinetic experiments performed with the nonhydrolyzable analogue ATPgammaS showed overall changes in fluorescence emission identical to those observed in the presence of ATP. In addition, the observed rate constants for the two fastest reaction phases were identical in ATP or ATPgammaS. The observed rate constant for the slowest phase showed a 4-fold reduction in the presence of ATPgammaS. Results in ATPgammaS using an alternate fluorophore labeling pattern suggest a third ternary intermediate may form prior to ssDNA product release. The existence of two or three ternary intermediates in strand exchange with a 30 bp duplex suggests the possibility that the step size for base pair switching may be 10-15 bp. Products of reactions in the presence of ATP and ATPgammaS, with and without proteinase K treatment, were analyzed on native polyacrylamide gels. In reactions in which only short-range RecA-DNA interactions were important, ATP hydrolysis was not required for recycling of RecA from both oligonucleotide products. Hydrolysis or deproteinization was required for RecA to release the heteroduplex product, but not the outgoing single strand.     

Unveiling two distinct ribonuclease activities and a topoisomerase activity in a site-specific DNA recombinase

The site-specific DNA recombinase Flp shows two types of RNA cleavage activities on hybrid DNA-RNA substrates. One targets the phosphodiester position involved in DNA recombination and follows a related mechanistic path. In this two-step reaction, first-strand scission is mediated by a nucleophilic attack of the scissile phosphodiester bond by the active site tyrosine of Flp. The resultant 3'-O-phosphoryl tyrosine bond is then attacked by the adjacent 2'-hydroxyl group. The second activity targets the immediately adjacent phosphodiester bond to the 3' side using a distinct mechanism. In this reaction, the vicinal 2'-hydroxyl directly attacks the phosphate group in a manner that is reminiscent of the pancreatic RNase mechanism. The Flp protein can also be shown to possess a topoisomerase-like activity.     

Mechanism of active site exclusion in a site-specific recombinase: role of the DNA substrate in conferring half-of-the-sites activity

The Flp site-specific recombinase assembles its active site by recruiting the catalytic tyrosine (Tyr-343) from one Flp monomer into the pro-active site containing a triad of Arg-191, His-305, and Arg-308 from a second monomer. In principle, two active sites may be assembled from a Flp dimer by simultaneous, reciprocal contribution of the shared amino acids by its constituent monomers. In practice, only one of the two active sites is assembled at a time, as would be consistent with a recombination mechanism involving two steps of single-strand exchanges. By using substrates containing strand-specific base bulges, we demonstrate that the relative disposition of their DNA arms can account for this active site exclusion. We also show that the exclusion mechanism operates only at the level of positioning Tyr-343 with respect to the pro-active site, and not at the level of orienting the labile phosphodiester bond within the DNA chain. It is not negative cooperativity of substrate binding but, rather, the substrate-induced negative cooperativity in protein orientation that accomplishes half-of-the-sites activity in the Flp system.     

Mutational analysis of the Mu transposase. Contributions of two distinct regions of domain II to recombination

Mu transposase is a member of a protein family that includes many transposases and the retroviral integrases. These recombinases catalyze the DNA cleavage and joining reactions essential for transpositional recombination. Here we demonstrate that, consistent with structural predictions, aspartate 336 of Mu transposase is required for catalysis of both DNA cleavage and DNA joining. This residue, although located 55 rather than 35 residues NH2-terminal of the essential glutamate, is undoubtedly the analog of the second aspartate of the Asp-Asp-35-Glu motif found in other family members. The core domain of Mu transposase consists of two subdomains: the NH2-terminal subdomain (IIA) contains the conserved Asp-Asp-Glu motif residues, whereas the smaller COOH-terminal subdomain (IIB) contains a large positively charged region exposed on its surface. To probe the function of domain IIB, we constructed mutant proteins carrying deletion or substitution mutations within this region. The activity of the deletion proteins revealed that domains IIA and IIB can be provided by different subunits in the transposase tetramer. Substitution mutations at two pairs of exposed lysine residues within the positively charged surface of domain IIB render transposase defective in transposition at a reaction step after DNA cleavage but prior to DNA joining. The severity of this defect depends on the structure of the DNA flanking the cleavage site. Thus, these data suggest that domain IIB is involved in manipulating the DNA near the cleavage site and that this function is important during the transition between the DNA cleavage and the DNA joining steps of recombination.     

Specific mismatch recognition in heteroduplex intermediates by p53 suggests a role in fidelity control of homologous recombination

We demonstrate that wild-type p53 inhibits homologous recombination. To analyze DNA substrate specificities in this process, we designed recombination experiments such that coinfection of simian virus 40 mutant pairs generated heteroduplexes with distinctly unpaired regions. DNA exchanges producing single C-T and A-G mismatches were inhibited four- to sixfold more effectively than DNA exchanges producing G-T and A-C single-base mispairings or unpaired regions of three base pairs comprising G-T/A-C mismatches. p53 bound specifically to three-stranded DNA substrates, mimicking early recombination intermediates. The KD values for the interactions of p53 with three-stranded substrates displaying differently paired and unpaired regions reflected the mismatch base specificities observed in recombination assays in a qualitative and quantitative manner. On the basis of these results, we would like to advance the hypothesis that p53, like classical mismatch repair factors, checks the fidelity of homologous recombination processes by specific mismatch recognition.     

Structural requirements of the higher order RNA kissing element in the enteroviral 3'UTR

The origin of replication ( oriR ) involved in the initiation of (-) strand enterovirus RNA synthesis is a quasi-globular multi-domain RNA structure which is maintained by a tertiary kissing interaction. The kissing interaction is formed by base pairing of complementary sequences within the predominant hairpin-loop structures of the enteroviral 3' untranslated region. In this report, we have fully characterised the kissing interaction. Site-directed mutations which affected the different base pairs involved in the kissing interaction were generated in an infectious coxsackie B3 virus cDNA clone. The kissing interaction appeared to consist of 6 bp. Distortion of the interaction by mispairing of each of the base pairs involved in this higher order RNA structure resulted in either temperature sensitive or lethal phenotypes. The nucleotide constitution of the base which gaps the major groove of the kissing domain was not relevant for virus growth. The reciprocal exchange of the complete sequence involved in the kissing resulted in a mutant virus with wild type virus growth characteristics arguing that the base pair constitution is of less importance for the initiation of (-) strand RNA synthesis than the existence of the tertiary structure itself.     

In vitro V(D)J recombination: signal joint formation

The first step of V(D)J recombination, specific cleavage at the recombination signal sequence (RSS), can be carried out by the recombination activating proteins RAG1 and RAG2. In vivo, the cleaved coding and signal ends must be rejoined to generate functional antigen receptors and maintain chromosomal integrity. We have investigated signal joint formation using deletion and inversion substrates in a cell free system. RAG1 and RAG2 alone or in combination were unable to generate signal joints. However, RAG1 and RAG2 complemented with nuclear extracts were able to recombine an extrachromosomal substrate and form precise signal joints. The in vitro reaction resembled authentic V(D)J recombination in being Ku-antigen-dependent.     

Failure of hairpin-ended and nicked DNA To activate DNA-dependent protein kinase: implications for V(D)J recombination

V(D)J recombination is initiated by a coordinated cleavage reaction that nicks DNA at two sites and then forms a hairpin coding end and blunt signal end at each site. Following cleavage, the DNA ends are joined by a process that is incompletely understood but nevertheless depends on DNA-dependent protein kinase (DNA-PK), which consists of Ku and a 460-kDa catalytic subunit (DNA-PKCS or p460). Ku directs DNA-PKCS to DNA ends to efficiently activate the kinase. In vivo, the mouse SCID mutation in DNA-PKCS disrupts joining of the hairpin coding ends but spares joining of the open signal ends. To better understand the mechanism of V(D)J recombination, we measured the activation of DNA-PK by the three DNA structures formed during the cleavage reaction: open ends, DNA nicks, and hairpin ends. Although open DNA ends strongly activated DNA-PK, nicked DNA substrates and hairpin-ended DNA did not. Therefore, even though efficient processing of hairpin coding ends requires DNA-PKCS, this may occur by activation of the kinase bound to the cogenerated open signal end rather than to the hairpin end itself.     

Direct interaction of aminopeptidase A with recombination site DNA in Xer site-specific recombination

Xer site-specific recombination at ColE1 cer converts plasmid multimers into monomers, thus ensuring the heritable stability of ColE1. Two related recombinase proteins, XerC and XerD, catalyse the strand exchange reaction at a 30 bp recombination core site. In addition, two accessory proteins, PepA and ArgR, are required for recombination at cer. These two accessory proteins are thought to act at 180 bp of accessory sequences adjacent to the cer recombination core to ensure that recombination only occurs between directly repeated sites on the same molecule. Here, we demonstrate that PepA and ArgR interact directly with cer, forming a complex in which the accessory sequences of two cer sites are interwrapped approximately three times in a right-handed fashion. We present a model for this synaptic complex, and propose that strand exchange can only occur after the formation of this complex.     

Reactivity of human apurinic/apyrimidinic endonuclease and Escherichia coli exonuclease III with bistranded abasic sites in DNA

Several oxidative DNA-damaging agents, including ionizing radiation, can generate multiply damaged sites in DNA. Among the postulated lesions are those with abasic sites located in close proximity on opposite strands. The repair of an abasic site requires strand scission by a repair endonuclease such as human apurinic/apyrimidinic endonuclease (Ape) or exonuclease III in Escherichia coli. Therefore, a potential consequence of the "repair" of bistranded abasic sites is the formation of double-strand breaks. To test this possibility and to investigate the influence of the relative distance between the two abasic sites and their orientation to each other, we prepared a series of oligonucleotide duplexes containing abasic sites at defined positions either directly opposite each other or separated by 1, 3, or 5 base pairs in the 5'- or 3'-direction. Analysis following Ape and exonuclease III treatment of these substrates indicated a variety of responses. In general, cleavage at abasic sites was slower in duplexes with paired lesions than in control duplexes with single lesions. Double-strand breaks were, however, readily generated in duplexes with abasic sites positioned 3' to each other. With the duplex containing abasic sites set 1 base pair apart, 5' to each other, both Ape and exonuclease III slowly cleaved the abasic site on one strand only and were unable to incise the other strand. With the duplex containing abasic sites set 3 base pairs apart, 5' to each other, Ape protein was unable to cleave either strand. These data suggest that closely positioned abasic sites could have several deleterious consequences in the cell. In addition, this approach has allowed us to map bases that make significant contact with the enzymes when acting on an abasic site on the opposite strand.     

Stimulation of intrachromosomal homologous recombination in human cells by electroporation with site-specific endonucleases

In somatic mammalian cells, homologous recombination is a rare event. To study the effects of chromosomal breaks on frequency of homologous recombination, site-specific endonucleases were introduced into human cells by electroporation. Cell lines with a partial duplication within the HPRT (hypoxanthine phosphoribosyltransferase) gene were created through gene targeting. Homologous intrachromosomal recombination between the repeated regions of the gene can reconstruct a functioning, wild-type gene. Treatment of these cells with the restriction endonuclease Xba I, which has a recognition site within the repeated region of HPRT homology, increased the frequency or homologous recombination bv more than 10-fold. Recombination frequency was similarly increased by treatment with the rare-cutting yeast endonuclease PI-Sce I when a cleavage site was placed within the repeated region of HPRT. In contrast, four restriction enzymes that cut at positions either outside of the repeated regions or between them produced no change in recombination frequency. The results suggest that homologous recombination between intrachromosomal repeats can be specifically initiated by a double-strand break occurring within regions of homology, consistent with the predictions of a model.