DNA cleavage in trans by the active site tyrosine during Flp recombination: switching protein partners before exchanging strands.

Each recombination event mediated by the Flp recombinase is the sum of four strand breakage and reunion reactions executed in two steps of two-strand exchanges. The reaction requires four Flp monomers. The key catalytic residue in Flp is Tyr-343. Arg-191, His-305, and Arg-308 appear to facilitate the cleavage and exchange steps of recombination. These four residues constitute the invariant tetrad of the Int family site-specific recombinases. Complementation tests between "step-arrest" mutants of Flp suggest that each Flp protomer harbors a "fractional active site." Hybrid "half site-recombinase" complexes reveal that efficient catalysis occurs when the Arg-His-Arg triad is present on one Flp monomer and the active site Tyr on a second monomer. Strand cleavage by an Flp monomer occurs virtually exclusively on the half site to which its partner protein is bound (cleavage in trans), and almost never on the half site to which it is bound (cleavage in cis). Trans-cleavage by Flp can provide a means for functionally exchanging Flp monomers between two DNA partners. Such a mechanism would be germane to recombination, since cleavage and rejoining in cis can only restore the parental substrate configuration and cannot yield recombinants.     

Functional analysis of box I mutations in yeast site-specific recombinases Flp and R: pairwise complementation with recombinase variants lacking the active-site tyrosine.

The site-specific recombinases Flp and R from Saccharomyces cerevisiae and Zygosaccharomyces rouxii, respectively, are related proteins that belong to the yeast family of site-specific recombinases. They share approximately 30% amino acid matches and exhibit a common reaction mechanism that appears to be conserved within the larger integrase family of site-specific recombinases. Two regions of the proteins, designated box I and box II, also harbor a significantly high degree of homology at the nucleotide sequence level. We have analyzed the properties of Flp and R variants carrying point mutations within the box I segment in substrate-binding, DNA cleavage, and full-site and half-site strand transfer reactions. All mutations abolish or seriously diminish recombinase function either at the substrate-binding step or at the catalytic steps of strand cleavage or strand transfer. Of particular interest are mutations of Arg-191 of Flp and R, residues which correspond to one of the two invariant arginine residues of the integrase family. These variant proteins bind substrate with affinities comparable to those of the corresponding wild-type recombinases. Among the binding-competent variants, only Flp(R191K) is capable of efficient substrate cleavage in a full recombination target. However, this protein does not cleave a half recombination site and fails to complete strand exchange in a full site. Strikingly, the Arg-191 mutants of Flp and R can be rescued in half-site strand transfer reactions by a second point mutant of the corresponding recombinase that lacks its active-site tyrosine (Tyr-343). Similarly, Flp and R variants of Cys-189 and Flp variants at Asp-194 and Asp-199 can also be complemented by the corresponding Tyr-343-to-phenylalanine recombinase mutant.     

Active-site assembly and mode of DNA cleavage by Flp recombinase during full-site recombination.

A combination of half-site substrates and step arrest mutants of Flp, a site-specific recombinase of the integrase family, had earlier revealed the following features of the half-site recombination reaction. (i) The Flp active site is assembled by sharing of catalytic residues from at least two monomers of the protein. (ii) A Flp monomer does not cleave the half site to which it is bound (DNA cleavage in cis); rather, it cleaves a half site bound by a second Flp monomer (DNA cleavage in trans). For the lambda integrase (Int protein), the prototype member of the Int family, catalytic complementation between two active-site mutants has been observed in reactions with a suicide attL substrate. By analogy with Flp, this observation is strongly suggestive of a shared active site and of trans DNA cleavage. However, reactions with linear suicide attB substrates and synthetic Holliday junctions are more compatible with cis than with trans DNA cleavage. These Int results either argue against a common mode of active-site assembly within the Int family or challenge the validity of Flp half sites as mimics of the normal full-site substrates. We devised a strategy to assay catalytic complementation between Flp monomers in full sites. We found that the full-site reaction follows the shared active-site paradigm and the trans mode of DNA cleavage. These results suggest that within the Int family, a unitary chemical mechanism of recombination is achieved by more than one mode of physical interaction among the recombinase monomers.     

Sequence of the loxP site determines the order of strand exchange by the Cre recombinase

Conservative site-specific recombinases of the integrase family carry out recombination via a Holliday intermediate. The Cre recombinase, a member of the integrase family, was previously shown to initiate recombination by cleaving and exchanging preferentially on the bottom strand of its loxP target sequence. We have confirmed this strand bias for an intermolecular recombination reaction that used wild-type loxP sites and Cre protein. We have examined the sequence determinants for this strand preference by selectively mutating the two asymmetric scissile base-pairs in the lox site (those immediately adjacent to the sites of cleavage by Cre). We found that the initial strand exchange occurs preferentially next to the scissile G residue. Resolution of the Holliday intermediate thus formed takes place preferentially next to the scissile A residue. Lys86, which contacts the scissile nucleotides in the Cre-lox crystal structures, was important for establishing the strand preference in the resolution of the loxP-Holliday intermediate, but not for the initiation of recombination between loxP sites.     

Active site electrostatics protect genome integrity by blocking abortive hydrolysis during DNA recombination

Water, acting as a rogue nucleophile, can disrupt transesterification steps of important phosphoryl transfer reactions in DNA and RNA. We have unveiled this risk, and identified safeguards instituted against it, during strand cleavage and joining by the tyrosine site‐specific recombinase Flp. Strand joining is threatened by a latent Flp endonuclease activity (type I) towards the 3′‐phosphotyrosyl intermediate resulting from strand cleavage. This risk is not alleviated by phosphate electrostatics; neutralizing the negative charge on the scissile phosphate through methylphosphonate (MeP) substitution does not stimulate type I endonuclease. Rather, protection derives from the architecture of the recombination synapse and conformational dynamics within it. Strand cleavage is protected against water by active site electrostatics. Replacement of the catalytic Arg‐308 of Flp by alanine, along with MeP substitution, elicits a second Flp endonuclease activity (type II) that directly targets the scissile phosphodiester bond in DNA. MeP substitution, combined with appropriate active site mutations, will be useful in revealing anti‐hydrolytic mechanisms engendered by systems that mediate DNA relaxation, DNA transposition, site‐specific recombination, telomere resolution, RNA splicing and retrohoming of mobile introns.     

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.     

Resolution of tethered antiparallel and parallel holliday junctions by the Flp site-specific recombinase

Members of the integrase family site-specific recombinases (also called the tyrosine family) bring about recombination in two steps by exchanging pairs of single strands at a time. The product of the first exchange reaction is a four-way DNA junction, the Holliday intermediate. The conformational dynamics by which the recombination complex "isomerizes" from the Holliday-forming to the Holliday-resolving mode are not well understood. Experiments with the lambda Int and Escherichia coli XerC/XerD systems imply that the strand configurations at the branch point of the protein-free junction dictate the resolution mode in the protein-bound junction. We have examined the question of strand bias during resolution for the Flp system by using a series of synthetic Holliday junctions that are conformationally constrained by local sequences or by strand tethering. We have not observed a strong resolution bias in favor of the strands designed to assume the "crossed" configuration within the unbound junction. The resolution patterns with antiparallel junctions in a variety of substrate contexts reveal either parity in strand choice, or only modest disparity. On the other hand, the highly biased resolutions observed in the case of tethered parallel junctions can be explained by the non-equivalence in protein occupancy of the DNA arms of these substrates and/or inefficient conversion of cleavage events to recombinants at the tethered ends.     

Peptide trapping of the Holliday junction intermediate in Cre-loxP site-specific recombination

Cre recombinase is a prototypical member of the tyrosine recombinase family of site-specific recombinases. Members of this family of enzymes catalyze recombination between specific DNA sequences by cleaving and exchanging one pair of strands between the two substrate sites to form a 4-way Holliday junction (HJ) intermediate and then resolve the HJ intermediate to recombinant products by a second round of strand exchanges. Recently, hexapeptide inhibitors have been described that are capable of blocking the second strand exchange step in the tyrosine recombinase recombination pathway, leading to an accumulation of the HJ intermediate. These peptides are active in the lambda-integrase, Cre recombinase, and Flp recombinase systems and are potentially important tools for both in vitro mechanistic studies and as in vivo probes of cellular function. Here we present biochemical and crystallographic data that support a model where the peptide inhibitor binds in the center of the recombinase-bound DNA junction and interacts with solvent-exposed bases near the junction branch point. Peptide binding induces large conformational changes in the DNA strands of the HJ intermediate, which affect the active site geometries in the recombinase subunits.     

Crystal structure of a Flp recombinase-Holliday junction complex: assembly of an active oligomer by helix swapping

The crystal structure of a Flp recombinase tetramer bound to a Holliday junction intermediate has been determined at 2.65 A resolution. Only one of Flp's two domains, containing the active site, is structurally related to other lambda integrase family site-specific recombinases, such as Cre. The Flp active site differs, however, in that the helix containing the nucleophilic tyrosine is domain swapped, such that it cuts its DNA target in trans. The Flp tetramer displays pseudo four-fold symmetry matching that of the square planar Holliday junction substrate. This tetramer is stabilized by additional novel trans interactions among monomers. The structure illustrates how mechanistic unity is maintained on a chemical level while allowing for substantial variation on the structural level within a family of enzymes.     

The isomeric preference of Holliday junctions influences resolution bias by lambda integrase.

Lambda site-specific recombination proceeds by a pair of sequential strand exchanges that first generate and then resolve a Holliday junction intermediate. A family of synthetic Holliday junctions with the branch point constrained to the center of the 7 bp overlap region was used to show that resolution of the top strands and resolution of the bottom strands are symmetrical but stereochemically distinct processes. Lambda integrase is sensitive to isomeric structure, preferentially resolving the pair of strands that are crossed in the protein-free Holliday junction. At the branch point of stacked immobile Holliday junctions, the number of purines is preferentially maximized in the crossed (versus continuous) strands if there is an inequality of purines between strands of opposite polarity. This stacking preference was used to anticipate the resolution bias of freely mobile junctions and thereby to reinforce the conclusions with monomobile junctions. The results provide a strong indication that in the complete recombination reaction a restacking of helices occurs between the top and bottom strand exchanges.     

Real-time single-molecule tethered particle motion analysis reveals mechanistic similarities and contrasts of Flp site-specific recombinase with Cre and λ Int

Flp, a tyrosine site-specific recombinase coded for by the selfish two micron plasmid of Saccharomyces cerevisiae, plays a central role in the maintenance of plasmid copy number. The Flp recombination system can be manipulated to bring about a variety of targeted DNA rearrangements in its native host and under non-native biological contexts. We have performed an exhaustive analysis of the Flp recombination pathway from start to finish by using single-molecule tethered particle motion (TPM). The recombination reaction is characterized by its early commitment and high efficiency, with only minor detraction from ‘non-productive’ and ‘wayward’ complexes. The recombination synapse is stabilized by strand cleavage, presumably by promoting the establishment of functional interfaces between adjacent Flp monomers. Formation of the Holliday junction intermediate poses a rate-limiting barrier to the overall reaction. Isomerization of the junction to the conformation favoring its resolution in the recombinant mode is not a slow step. Consistent with the completion of nearly every initiated reaction, the chemical steps of strand cleavage and exchange are not reversible during a recombination event. Our findings demonstrate similarities and differences between Flp and the mechanistically related recombinases λ Int and Cre. The commitment and directionality of Flp recombination revealed by TPM is consistent with the physiological role of Flp in amplifying plasmid DNA.     

Half-site recombinations mediated by yeast site-specific recombinases Flp and R.

The Flp recombinase of Saccharomyces cerevisae and the related R recombinase of Zygosaccharomyces rouxii can efficiently catalyze strand cleavage and strand exchange reactions in half recombination sites. A half-site consists of one recombinase binding element, a recombinase cleavage site on one strand and a 5' spacer hydroxyl group on the other that can initiate the strand exchange reaction. We have studied the various types of strand exchanges that half-sites can participate in. Reaction between a left half-site and a right half-site generates a full recombination site. Strand transfer between two left half-sites or between two right half-sites produces pseudo-full-sites. Strand transfer within a half-site results in a stem-loop or hairpin product. The half-site strand transfer reaction is fairly indifferent to the spacer sequence of the substrate per se and is less sensitive to variations in spacer lengths than a full-site recombination reaction. The optimal spacer length of eight to ten nucleotides observed for the Flp half-site reaction likely permits the most productive catalytic interactions between two Flp monomers bound to each of two partner half-sites. When reacted with a full-site, the half-site can give rise to a normal or reverse recombinant, corresponding to homologous or non-homologous alignments of the spacer sequences during substrate synapsis. The contrary recombination (resulting from non-homologous spacer alignment), whose level is low relative to normal recombination, is partly suppressed when the half-site spacer ends in a 5'-phosphate rather than a 5'-hydroxyl group. Thus, the early steps of recombination, namely synapsis and initial stand transfer, are not dependent on complete spacer homology between the two recombining substrates. The selection of properly aligned substrate partners must occur at the homology dependent branch migration step. In reactions containing a mixture of Flp and R half-sites, Flp and R catalyze strand transfer, almost exclusively, within or between their respective cognate substrates. However, under conditions where self-crosses are inhibited, strand exchange between a Flp half-site and an R half-site appears to be stimulated by a combination of R and Flp.     

Dissecting the resolution reaction of lambda integrase using suicide Holliday junction substrates.

A reciprocal strand exchange between two DNA helices generates the crossed-strand intermediate, or Holliday junction, which is common to many pathways of homologous and site-specific recombination. The Int family of recombinases are unique in their ability to both make and resolve Holliday junctions. Previous experiments utilizing 'synthetic' att site Holliday junctions to study the mechanisms associated with the cleavage, transfer and ligation of DNA strands have been confined to studying reciprocal strand exchanges (a pair of temporally overlapping strand cleavages). To circumvent this limitation, we have designed synthetic suicide Holliday junctions that make it possible to monitor individual DNA strand cleavage events. These substrates contain a pre-existing nick in the vicinity of the Int binding site; when Int introduces a second nick into these substrates, the 5'OH nucleophile required for ligation (in either the forward or reverse reaction) is lost by diffusion, thus trapping the covalent protein-DNA intermediate. The results indicate that resolution (involving two partner Ints) is stimulated by additional 'cross-core' Ints as a result of enhanced cleavage rates, and not as a result of enhanced co-ordination of cleavage. Several models for the role of the 'cross-core' Ints during resolution are discussed, as well as the usefulness of these substrates for studying additional aspects of the Holliday junction resolution reaction.     

Xer site-specific recombination in vitro.

Two related recombinases, XerC and XerD, belonging to the lambda integrase family of enzymes, are required for Xer site-specific recombination in vivo. In order to understand the roles of these proteins in the overall reaction mechanism, an in vitro recombination system using a synthetic Holliday junction-containing substrate has been developed. Recombination of this substrate is efficient and requires both XerC and XerD. However, only exchange of one pair of strands, the one corresponding to the conversion of the Holliday junction intermediate back to the substrate, has been observed. Recombination reactions using XerC and XerD derivatives that are mutant in their presumptive catalytic residues, or are maltose-binding fusion recombinase derivatives, have demonstrated that this pair of strand exchanges is catalysed by XerC. The site of XerC-mediated cleavage has been located to between the last nucleotide of the XerC binding site and the first nucleotide of the central region. Cleavage at this site generates a free 5'-OH and a covalent complex between XerC and the 3' end of the DNA.     

Reactions of Cre with Methylphosphonate DNA: Similarities and Contrasts with Flp and Vaccinia Topoisomerase

Background

Reactions of vaccinia topoisomerase and the tyrosine site-specific recombinase Flp with methylphosphonate (MeP) substituted DNA substrates, have provided important insights into the electrostatic features of the strand cleavage and strand joining steps catalyzed by them. A conserved arginine residue in the catalytic pentad, Arg-223 in topoisomerase and Arg-308 in Flp, is not essential for stabilizing the MeP transition state. Topoisomerase or its R223A variant promotes cleavage of the MeP bond by the active site nucleophile Tyr-274, followed by the rapid hydrolysis of the MeP-tyrosyl intermediate. Flp(R308A), but not wild type Flp, mediates direct hydrolysis of the activated MeP bond. These findings are consistent with a potential role for phosphate electrostatics and active site electrostatics in protecting DNA relaxation and site-specific recombination, respectively, against abortive hydrolysis.

Methodology/Principal Findings

We have examined the effects of DNA containing MeP substitution in the Flp related Cre recombination system. Neutralizing the negative charge at the scissile position does not render the tyrosyl intermediate formed by Cre susceptible to rapid hydrolysis. Furthermore, combining the active site R292A mutation in Cre (equivalent to the R223A and R308A mutations in topoisomerase and Flp, respectively) with MeP substitution does not lead to direct hydrolysis of the scissile MeP bond in DNA. Whereas Cre follows the topoisomerase paradigm during the strand cleavage step, it follows the Flp paradigm during the strand joining step.

Conclusions/Significance

Collectively, the Cre, Flp and topoisomerase results highlight the contribution of conserved electrostatic complementarity between substrate and active site towards transition state stabilization during site-specific recombination and DNA relaxation. They have potential implications for how transesterification reactions in nucleic acids are protected against undesirable abortive side reactions. Such protective mechanisms are significant, given the very real threat of hydrolytic genome damage or disruption of RNA processing due to the cellular abundance and nucleophilicity of water.     

Electrostatic Suppression Allows Tyrosine Site-specific Recombination in the Absence of a Conserved Catalytic Arginine

The active site of the tyrosine family site-specific recombinase Flp contains a conserved catalytic pentad that includes two arginine residues, Arg-191 and Arg-308. Both arginines are essential for the transesterification steps of strand cleavage and strand joining in DNA substrates containing a phosphate group at the scissile position. During strand cleavage, the active site tyrosine supplies the nucleophile to form a covalent 3′-phosphotyrosyl intermediate. The 5′-hydroxyl group produced by cleavage provides the nucleophile to re-form a 3′-5′ phosphodiester bond in a recombinant DNA strand. In previous work we showed that substitution of the scissile phosphate (P) by the charge neutral methylphosphonate (MeP) makes Arg-308 dispensable during the catalytic activation of the MeP diester bond. However, in the Flp(R308A) reaction, water out-competes the tyrosine nucleophile (Tyr-343) to cause direct hydrolysis of the MeP diester bond. We now report that for MeP activation Arg-191 is also not required. In contrast to Flp(R308A), Flp(R191A) primarily mediates normal cleavage by Tyr-343 but also exhibits a weaker direct hydrolytic activity. The cleaved MeP-tyrosyl intermediate formed by Flp(R191A) can be targeted for nucleophilic attack by a 5′-hydroxyl or water and channeled toward strand joining or hydrolysis, respectively. In collaboration with wild type Flp, Flp(R191A) promotes strand exchange between MeP- and P-DNA partners. Loss of a catalytically crucial positively charged side chain can thus be suppressed by a compensatory modification in the DNA substrate that neutralizes the negative charge on the scissile phosphate.     

DNA recognition, strand selectivity, and cleavage mode during integrase family site-specific recombination

We have probed the association of Flp recombinase with its DNA target using protein footprinting assays. The results are consistent with the domain organization of the Flp protein and with the general features of the protein-DNA interactions revealed by the crystal structures of the recombination intermediates formed by Cre, the Flp-related recombinase. The similarity in the organization of the Flp and Cre target sites and in their recognition by the respective recombinases implies that the overall DNA-protein geometry during strand cleavage in the two systems must also be similar. Within the functional recombinase dimer, it is the interaction between two recombinase monomers bound on either side of the strand exchange region (or spacer) that provides the allosteric activation of a single active site. Whereas Cre utilizes the cleavage nucleophile (the active site tyrosine) in cis, Flp utilizes it in trans (one monomer donating the tyrosine to its partner). By using synthetic Cre and Flp DNA substrates that are geometrically restricted in similar ways, we have mapped the positioning of the active and inactive tyrosine residues during cis and trans cleavage events. We find that, for a fixed substrate geometry, Flp and Cre cleave the labile phosphodiester bond at the same spacer end, not at opposite ends. Our results provide a model that accommodates local heterogeneities in peptide orientations in the two systems while preserving the global functional architecture of the reaction complex.     

The Effect of Attachment Site Mutations on Strand Exchange in Bacteriophage λ Site-Specific Recombination

Recombination of phage λ attachment sites occurs by sequential exchange of the DNA strands at two specific locations. The first exchange produces a Holliday structure, and the second resolves it to recombinant products. Heterology for base substitution mutations in the region between the two strand exchange points (the overlap region) reduces recombination; some mutations inhibit the accumulation of Holliday structures, others inhibit their resolution to recombinant products. To see if heterology also alters the location of the strand exchange points, we determined the segregation pattern of three single and one multiple base pair substitution mutations of the overlap region in crosses with wild type sites. The mutations are known to differ in the severity of their recombination defect and in the stage of strand exchange they affect. The three single mutations behaved similarly: each segregated into both products of recombination,, and the two products of a single crossover were frequently nonreciprocal in the overlap region. In contrast, the multiple mutation preferentially segregated into one of the two recombinant products, and the two products of a single crossover appeared to be fully reciprocal. The simplest explanation of the segregation pattern of the single mutations is that strand exchanges occur at the normal locations to produce recombinants with mismatched base pairs that are frequently repaired. The segregation pattern of the multiple mutation is consistent with the view that both strand exchanges usually occur to one side of the mutant site. We suggest that the segregation pattern of a particular mutation is determined by which stage of strand exchange it inhibits and by the severity of the inhibition.     

Mutations of the phage lambda attachment site alter the directionality of resolution of Holliday structures.

Integrative recombination of bacteriophage lambda occurs by two sequential, reciprocal strand exchanges at specific positions within the attachment sites. Both exchanges are promoted by the lambda Int protein; the first forms a Holliday structure, and the second resolves it to recombinant products. Recombination requires sequence homology within the 7 bp 'overlap' region that separates the two points of strand exchange. To see if homology promotes the second strand exchange, we constructed attachment site Holliday structures by annealing DNA strands and then assayed Int-promoted resolution. Holliday structures corresponding to strand exchange between sites with homologous overlap regions were efficiently resolved to give mixtures of recombinants and parents. Holliday structures corresponding to exchanges between heterologous sites fell into two classes. Members of the first class, in which heterology limited but did not completely prevent migration of the branchpoint within the overlap region, were resolved efficiently and preferentially to parental molecules. We propose that resolution to recombinants occurs only if homology allows branch migration from the first to the second exchange site. Members of the second class, in which heterology constrained the branchpoint within an Int binding site, were resolved poorly. We suggest that Holliday structures that have a branchpoint within an Int binding site are poor substrates for Int.     

Alterations in the directionality of lambda site-specific recombination catalyzed by mutant integrases in vivo.

Phage lambda integrative and excisive recombination normally proceeds by a pair of sequential strand exchanges. During the first exchange reaction, the "top" strand in each recombination site is cleaved, exchanged, and religated generating a Holliday junction intermediate. This intermediate DNA structure is resolved through a pair of reciprocal "bottom" strand exchanges, leading to recombinant products. The strict co-ordination of exchange reactions ensures religation between correct partner strands only. Here we show that the directionality of recombination is altered in vivo by two mutant integrases, Int-h (E174 K) and a double mutant Int-h/218 (E174 K/E218 K). This change in directionality leads to deletion instead of inversion on substrates that carry inverted attachment sites and, depending on the pair of target sites employed, requires the presence or absence of integration host factor. Neither Fis nor Xis is involved in deletion. Sequence analyses of deletion products reveal that the newly generated hybrid attachment site exhibits a reversed genetic polarity. We demonstrate that only one of two possible hybrid site configurations is generated and discuss two pathways leading to deletion. In the first, deletion results from a wrong alignment of the two recombination sites within the synaptic complex. In the second pathway, the unco-ordinated cleavage by the mutant integrases of all four DNA strands present in a conventional Holliday junction intermediate leads to two double-stranded breaks, whereby the subsequent rejoining between "wrong" partner strands appears restricted to only two strands.