©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Role of Partner Homology in DNA Recombination
COMPLEMENTARY BASE PAIRING ORIENTS THE 5`-HYDROXYL FOR STRAND JOINING DURING Flp SITE-SPECIFIC RECOMBINATION (*)

(Received for publication, October 12, 1994; and in revised form, December 16, 1994)

Joonsoo Lee Makkuni Jayaram (§)

From the Department of Microbiology, University of Texas at Austin, Austin, Texas 78712

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Absolute homology between partner substrates within the strand exchange region is an essential requirement for recombination mediated by the yeast site-specific recombinase Flp. Using combinations of specially designed half- and full-site Flp substrates, we demonstrate that the strand joining step of recombination is exquisitely sensitive to spacer homology. At each exchange point, 2-3 spacer nucleotides adjacent to the nick within the cleaved strand of one substrate must base pair with the corresponding segment of the un-nicked strand from the second substrate for efficient strand joining in the recombinant mode. In accordance with the ``cis-activation/trans-nucleophilic attack'' model for each of the two transesterification steps of Flp recombination (strand cleavage and strand joining), we propose that the limited strand pairing orients the DNA-nucleophile (5`-hydroxyl) for attack on its target diester (3`-phosphotyrosyl-Flp). During one round of recombination, 4-6 terminal base pairs of the spacer (2-3 base pairs at each spacer end) must unpair, following strand cleavage, within a DNA substrate and pair with the partner substrate prior to strand union. In this model, the extent of branch migration of the covalently closed Holliday intermediate is limited to the central core of the spacer. The templated positioning of reactive nucleic acid groups (which is central to the model) may be utilized by other recombination systems and by RNA splicing reactions.


INTRODUCTION

Models for general recombination postulate an early strand invasion step (initiation) followed by formation of a Holliday junction, extension of heteroduplex DNA (propagation), and production of mature recombinants by resolution of the Holliday junction (termination) (reviewed in Radding(1991), West(1992), and Kowalczykowski(1994)). The reaction requires the action of several proteins and may span DNA segments that could be several thousand bp (^1)long. The steps involved in site-specific recombination mediated by the integrase (the Int protein of phage ) family recombinases (Argos et al., 1986; Abremski and Hoess, 1992) have parallels to those involved in general recombination. The reaction, though, is miniaturized dramatically. Four protein monomers, with or without the help of a limited number of accessory protein factors, carry out the reaction. The propagation of ``heteroduplex'' during Int family recombination is confined to a short DNA segment (less than 10 bp) called the strand exchange region (also referred to as the spacer or overlap region). The reaction proceeds via formation and subsequent resolution of a Holliday intermediate. The overall biochemical similarities between the two recombination pathways suggest that at least some of their steps may share common mechanistic features as well.

Recombination catalyzed by three site-specific recombinases of the Int family, Int, the Flp protein from Saccharomyces cerevisiae, and the Cre protein from phage P1, is dependent on absolute homology between partner substrates within the region of strand exchange (Craig, 1988; Sadowski, 1993). In a general sense, homology may influence the reaction at a prechemical step (perhaps synapsis) or the chemical steps (strand cleavage and strand union) or a post-chemical step (branch migration of the Holliday junction resulting from the first pair of strand exchanges).

The Int reaction has been extensively explored with regard to its requirement for homology between recombination partners. In order to obtain efficient reaction between normal homoduplex attP (phage attachment site) and attB (bacterial attachment site) partners, their overlap regions need to be perfectly homologous (Weisberg et al., 1983; Bauer et al., 1985). However, in a cross between a homoduplex attP and a heteroduplex attB, normal recombination frequency is realized if either of the two attB strands is homologous to attP (Bauer et al., 1985). An incisive account of the several models proposed to account for this homology requirement and the experimental tests of their validity is given in Cowart et al.(1991). The most significant observation is that spacer heterology positioned away from the site of the first exchange has little effect on initiation of recombination, namely cleavage and exchange of top strands (Nunes-Duby et al., 1989); apparently, so does a similarly placed cross-link within the spacer (Cowart et al., 1991). However, either manipulation invariably blocks the completion of the event, namely, cleavage and exchange of the bottom strands (Bauer et al., 1984; Kitts and Nash, 1987; Nunes-Duby et al., 1987; Cowart et al., 1991). The simplest explanation is that branch migration of the Holliday junction across the overlap segment precedes its resolution; nonhomology impedes branch migration and consequently inhibits the second exchange. The yield of the Holliday intermediate is dependent on the position of heterology. When the heterology is immediately adjacent to the point of top strand exchange, Holliday junctions are not detected. Presumably, a junction blocked in the immediate vicinity of the first exchange point is resolved to the parental strands by a reversal of the exchange reaction.

As pointed out earlier, the Flp site-specific recombinase (which plays a central role in the copy number control of the yeast plasmid 2 µm circle; reviewed in Broach and Volkert(1991)) is a member of the Int family. Homology of the strand exchange region between partner DNA substrates is an essential feature of the Flp-mediated recombination reaction as well (Senecoff and Cox, 1986). Point mutations within the spacer sequence of one substrate can be rescued by identical mutations within the second substrate. Furthermore, in half-site to full-site crosses, the strand transfer product derived from the homologous (normal) alignment of the sites far exceeds that derived from the nonhomologous (reverse) alignment (Serre et al., 1992). Similar results were also obtained by Qian et al.(1992). They noted that the mode of productive alignment of the half-site with the full site was determined by homology of the 2 base pairs immediately adjacent to the exchange point, the cleavage-distal base pair being the more critical of the two. Spacer nonhomology at this position did not lead to accumulation of a semi-Holliday or ``Y'' structure expected to result from a single exchange event between a half-site and its full-site partner (see Fig. 1, B and C). Qian et al.(1992) surmised that homology is sensed at an early step of the recombination pathway.


Figure 1: Recombination reactions with half-site substrates. The half-site and full-site substrates are schematically shown with their spacer positions numbered. The parallel arrows represent the binding element for an Flp monomer. The positions within the half-site spacer marked X and N refer to those bases that have been altered in a number of experiments to produce heterologies between substrate partners. The asterisk indicates the labeled 5` end by which the recombination products can be monitored. A, cleavage of the half-site results in the loss of the dinucleotide (5`-HOXX-3`) by diffusion. Intramolecular strand union results in a hairpin product. Strand transfer between two half-sites (inter-half-site recombination) is also possible. In our denaturing gel electrophoresis assay, this product is indistinguishable from the hairpin as both contain the same radioactive strand. B and C, cleavage within a half-site and its full-site partner followed by intermolecular strand transfer produces the indicated recombinant strands. In B the half-site follows the ``left alignment,'' recombining with the top strand of the full site. In C the half-site follows the ``right alignment,'' recombining with the bottom strand of the full site.



Does the Int paradigm for the role of homology in recombination apply globally to members of the Int family? Here we address this issue in the Flp recombination reaction using half-site and full-site substrates. We conclude that the first functional role of homology is in aligning the 5`-hydroxyl of a nicked DNA strand using Watson-Crick base pairing during the strand joining reaction. The result is consistent with a model in which homology-dependent branch migration of the Holliday intermediate may be limited essentially to the central 2-4-base pair region of the spacer sequence.


MATERIALS AND METHODS

Purification of Flp

All reactions were done with wild-type Flp. The protein was partially purified essentially as described by Prasad et al.(1987). Further purification was achieved by chromatography on an affinity column containing oligomeric Flp site oligodeoxynucleotides linked to CNBr-activated Sepharose (Parsons et al., 1990). Purity of the final Flp preparation was approximately 90%. Flp protein concentrations were estimated by comparisons between densitometric scans of Coomassie Brilliant Blue-stained Flp aliquots (fractionated in SDS-polyacrylamide gels) and similar scans of known amounts of bovine serum albumin.

Synthetic Half and Full Recombination Sites and Holliday Junctions

Oligodeoxynucleotides for construction of half-sites, full sites, and Holliday junctions were synthesized in an Applied Biosystems DNA synthesizer (model 380A) using phosphoramidite chemistry (Beaucage and Caruthers, 1981). Approximately 2-10 pmol each of the appropriate oligodeoxynucleotides were mixed in TE (10 mM Tris-HCl, pH 7.8 at 23 °C; 1 mM EDTA, pH 8.0), heated to 65 °C for 10 min, and cooled slowly to room temperature. Normally the radioactively labeled strand was held at one-half to one-third the concentration of the unlabeled partner strands during hybridization. The synthetic Holliday junctions and some of the linear substrates were gel-purified prior to being used in strand cleavage or strand transfer assays. The relevant features of the synthetic substrates are described under ``Results'' and displayed in the figures. The complete sequences of the substrates are available upon request.

To label the 5` end of a deoxyoligonucleotide, it was phosphorylated by T4 polynucleotide kinase in presence of [-P]ATP. In some cases, the 5`-hydroxyl group was phosphorylated with non-radioactive ATP. The 3` end was labeled with alpha-P-labeled cordycepin phosphate. The unreacted ATP or cordycepin phosphate was removed by spin dialysis on a G-25 column. Hybridization to the partner oligodeoxynucleotide(s) was done in TE.

Strand Transfer Reactions in Half-sites

The conditions for half-site strand transfer were essentially the same as those described previously (Chen et al., 1992). Normally the reactions contained approximately 0.02-0.05 pmol of the half-site and an excess of Flp (8-10 pmol of Flp/pmol of Flp binding element) in a reaction volume of 30 µl. Reactions were terminated by the addition of SDS (0.1% final concentration) and treated with proteinase K (100 µg/reaction for 1 h at 37 °C). After chloroform-phenol extraction and ethanol precipitation, the DNA was fractionated by electrophoresis in 10% denaturing polyacrylamide gels (acrylamide:bisacrylamide, 19:1). Since the reactions contained a radioactively labeled substrate, the strand exchange products were visualized by autoradiography.

Recombination between a Half-site and a Full Site

In these reactions, the half-site was labeled at the 5` end on the cleavage strand. The unlabeled full-site partner was an approximately 100-bp DNA fragment that contained one copy of the Flp recombination site. Reactions contained approximately 0.02-0.05 pmol of the half-site, 0.1-0.2 pmol of the full site, and 2 pmol of Flp in an incubation volume of 30 µl. Reactions were done as described for half-site strand transfer.

Strand Transfer between Synthetic Full Sites

The synthetic full sites (approximately 45-50 bp long carrying EcoRI and HindIII overhangs at the ends) were poor substrates in strand transfer. To increase the efficiency of the reaction, assays were done with the monomeric form of a radioactively labeled full site and the concatemeric form of the unlabeled full-site partner. The concatemer was prepared as follows. A mixture containing the phosphorylated and unphosphorylated form of the top strand in 1:4 molar ratio was hybridized to the fully phosphorylated bottom strand. This mixture was ligated at room temperature for 2 h under conditions that gave concatemers containing, on an average, 3 units of the monomer. The strand transfer reactions were carried out using the normal protocols described for half-site and half-site to full-site recombinations. The ratio of the labeled substrate to the monomeric equivalent of the unlabeled substrate was roughly 1:10. Approximately 6-8 pmol of Flp/pmol of Flp binding element were present in these reactions carried out in 30-µl incubation mixtures.

Strand Cleavage Assays

The strand cleavage reactions were done under strand transfer conditions using approximately 0.01 pmol of the labeled substrate in a 30-µl reaction mixture (Chen et al., 1992). Normally, the assays contained six to eight monomers of Flp monomer/Flp binding element. In some assays, a range of Flp to substrate molar ratios was employed. Reactions were stopped at various times by immersing samples in a boiling water bath for 5 min. In those instances where the labeled cleavage product was less than 10 bp long, aliquots of the quenched reaction mixtures were directly fractionated in 15 or 20% denaturing polyacrylamide gels (acrylamide:bisacrylamide, 19:1). Otherwise samples were made 0.1% in SDS (w/v), treated with proteinase K (100 µg/sample for 1 h at 37 °C), and were further processed as described for the strand transfer reactions. The cleavage product was separated from the substrate by electrophoresis in 10% denaturing polyacrylamide gels.

Full-site-assisted Cleavage in Half-sites

In these assays the molar ratio of the labeled half-site to Flp was deliberately decreased to inhibit cleavage. A typical reaction contained approximately 0.002-0.004 pmol of the 3` end-labeled half-site and 0. 2 pmol of Flp in a volume of 60 µl. The basal cleavage under this reaction condition was then stimulated by the addition of approximately 0.05 to 0.1 pmol of the unlabeled full site. Samples were processed as described for the regular cleavage assays.

Assay for Strand Transfer from a Precleaved Substrate

The substrate for assaying the strand joining reaction was prepared as follows. Approximately 0.5 pmol of a half-site labeled at the 5` end on the cleavage strand and phosphorylated at the spacer terminus on the bottom strand (to prevent strand joining) was treated with Flp under normal recombination conditions to cleave the DNA. After two cleavage cycles, the reaction was treated with proteinase K and processed as detailed under the strand transfer assays. Following electrophoresis in a 10% denaturing polyacrylamide gel, the labeled band corresponding to the cleaved DNA linked to the Flp peptide was excised and eluted. The DNA was then hybridized to a 2-fold molar excess of the two deoxyoligonucleotides that complete the assembly of the strand transfer substrate. The longer of the latter two deoxyoligonucleotides forms the bottom strand of the substrate; the shorter one anneals adjacent to the cleavage point and serves as the phosphoryl acceptor. Strand joining assays were done under standard conditions for strand transfer in half-sites.

General Methods

Restriction enzyme digestions, isolation of plasmid DNA and other miscellaneous procedures were done as described by Maniatis et al.(1982).


RESULTS

Flp half-site substrates (Amin et al., 1991; Qian et al., 1992; Serre et al., 1992) modeled after the half-att sites (Nunes-Duby et al., 1989) have been instrumental in revealing several aspects of the mechanism of Flp recombination (Chen et al., 1992; Lee and Jayaram, 1993; Pan et al., 1993). A half-site (see Fig. 1) contains one Flp binding element, one cleavage site, and one strand transfer-competent 5`-hydroxyl group. Hence it can undergo one strand cleavage and one strand joining. A half-site may partake in an intramolecular recombination event (Fig. 1A) or an intermolecular recombination event with a full site (Fig. 1, B and C) or with a second half-site (not shown in Fig. 1). Depending on the mode of alignment, the half-site to full-site reaction yields an exchange product with the top strand (left alignment; Fig. 1B) or with the bottom strand of the full site (right alignment; Fig. 1C). We reasoned that the nature of recombinant products from reactions containing half-sites with built-in alterations in the spacer region together with full sites harboring the normal spacer or spacer variants might provide clues to the homology-dependent step(s) of recombination.

The strand cleavage and joining steps of Flp recombination are transesterification reactions. The cleavage reaction is executed via nucleophilic attack by the active site tyrosine of Flp (Tyr-343) on the scissile phosphoester bond within DNA. Cleavage produces a 3`-phosphotyrosyl bond between DNA and Flp plus a 5`-hydroxyl group. The joining reaction is mediated via nucleophilic attack by the 5`-hydroxyl group from a nicked DNA substrate on the phosphotyrosyl bond formed within the partner substrate. Since the target diester in the cleavage step and the active nucleophile in the strand joining step are part of the DNA substrate, it seemed plausible that base complementarity could be utilized to position them within the reaction center. Lack of base pairing could then affect either step by misalignment of these reactive groups. The experiments compiled here focus predominantly on the effect of noncomplementarity at specific spacer positions on the strand joining reaction.

Spacer Homology between Partners Is Not Tested at the Strand Cleavage Step

As alluded to earlier, homology between the spacers of partner substrates provides the basis for partner selectivity in the Flp reaction. Does Flp discriminate between homologous and nonhomologous target sites prior to initiating the chemical steps of recombination? Two half-site substrates used in this test differed only in spacer positions 1 and 2 (5`-AC-3`/3`-TG-5` and 5`-CT-3`/3`-GA-5`, Fig. 2) and were cleavage-competent. In the assay conditions, the concentration of the half-site (3` end-labeled on the cleavage strand) was decreased while maintaining a high Flp monomer to half-site molar ratio (approximately 50:1 or greater) until strand cleavage dropped to very low levels (lane 2 of Fig. 2, A and B). It is known that cleavage of a Flp-bound half-site requires its association with a second Flp-bound half-site (Chen et al., 1993). This is consistent with the shared active site model for Flp (Chen et al., 1992, 1993) in which a monomer of Flp acquires Tyr-343 from a second Flp monomer to execute strand cleavage. At the low half-site concentrations employed here, productive association between Flp-bound half-sites may become rate-limiting. Addition of an optimal concentration of the unlabeled full site to the reaction mixture was then found to restore cleavage levels to normal (lanes 3 and 4 of Fig. 2, A and B). It is likely that cleavage could now occur within the half-site via synapsis between the half-site and the full site. Alternatively, cooperative protein-DNA and protein-protein interactions might assist the assembly of a synaptic complex between the full-site and a noncovalent dimeric form of the half-site (a pseudo-full site). The half-site may then be cleaved within this complex. In either case, the relevant result is that the degree of cleavage stimulation was the same whether or not the full site and half-site partners were homologous at spacer positions 1 and 2 (compare lane 3 against lane 4 of Fig. 2, A and B).


Figure 2: Full-site-assisted cleavage of half-sites. The half-site substrate used in each reaction set is shown at the top. The half-site was labeled on the cleavage strand at the 3` end (A, B) or at the 5` end (C, D). The two full-site partner substrates used are also indicated. The substrate and cleavage products are denoted by S and CL, respectively. The half-site strand transfer product is marked HP; the product of exchange between the half-site and a full site is indicated by FP.



The same assay was duplicated using the half-sites labeled at the 5` end of the cleavage strands so that formation of strand transfer products could be monitored. Stimulation of half-site cleavage by the full sites could be indirectly gleaned as the increase in the half-site recombinant product (HP; compare lane 2 against lane 3 and 4 of Fig. 2, C and D). Note, however, that the half-site to full-site recombinant was detectable only between the homologous pair (FP; lane 3 of Fig. 2C; lane 4 of Fig. 2D) and not between the nonhomologous pair (lane 4 of Fig. 2C; lane 3 of Fig. 2D). The strong implication of this result, together with that depicted in Fig. 2, A and B, is that spacer homology at positions 1 and 2 between partners is sensed following strand cleavage, not prior to it.

Spacer Mismatches at Positions 1-3, but Not beyond, Affect Strand Joining in Half-sites

Earlier experiments (data not shown) implied that, given the same level of cleavage, strand joining in a half-site reaction is limited by the pairing potential between spacer positions 1-3 and 1`-3`. During strand union within a half-site by spacer looping or between two half-sites by spacer overlay, positions 1`-3` would be situated in a possible base pairing context with respect to positions 1-3. To verify the significance of this complementarity, a pair of half-sites with TT/AA at spacer positions 1 and 2 were constructed (Fig. 3). The sequences of their bottom strand spacers contained an A at position 3, but differed at positions 4 and 1`-4`. In one half-site, 1-3 and 1`-3` were perfectly complementary; in the other, these positions were mismatched. The kinetics of strand cleavage and strand transfer were followed for each substrate by placing the P label at the 3` end and 5` end, respectively, on the cleavage strands. As expected, the two substrates showed virtually identical cleavage kinetics (Fig. 3, A and B); the kinetics of strand transfer, though, were vastly different (Fig. 3, C and D). For equivalent cleavage events, therefore, 1-3 to 1`-3` complementarity determines the rate of strand joining. However, at longer time intervals substantial strand joining was seen even in the absence of the 1-3 to 1`-3` complementarity. Since the half-site reaction is predominantly intramolecular, it is not surprising that nonhomology did not affect the final product yield, although it was delayed significantly. The slow rate of recombinant formation with the substrate that cannot form 1-3 to 1`-3` match was not due to the reversal of the joining reaction. The hairpin product of recombination, once formed, was refractory to cleavage by Flp. When this product was chemically synthesized and incubated with Flp, very little cleavage was obtained (data not shown).


Figure 3: Effect of spacer sequence distal to the reaction center on strand cleavage and strand joining in half-sites. The two half-sites used in the assays are shown. They were labeled at the 3` end for the cleavage assays (A, B) and at the 5` end for the strand transfer assays (C, D). The half-site in the reactions displayed in A and C can produce a perfect match between spacer positions 1-3 and 1`-3` on the noncleavage strand. These positions are noncomplementary for the half-site in the reactions represented by B and D. Strand cleavage and strand transfer were assayed at the time points indicated. The bands corresponding to substrate, cleavage product and strand transfer product are marked S, CL, and P, respectively.



In Flp recombination, the strand cleavage and strand joining reactions can be uncoupled by using appropriate substrates. A half-site linked at the exchange phosphate to a short Flp peptide via Tyr-343 or to tyramine can take part in the strand joining reaction when supplied with Flp or a strand transfer-competent Flp variant (Pan and Sadowski, 1992; Lee and Jayaram, 1993). A set of substrates for monitoring strand joining was assembled such that three mismatches were placed right next to the point of strand joining (positions 1-3) and 3 (positions 4-6) and 6 (positions 7-9) base pairs downstream of it (Fig. 4). The yield of the joined molecule was the same for the fully matched (lane 2, Fig. 4), 4-6 mismatched (lane 4; Fig. 4) and 7-9 mismatched (lane 5, Fig. 4) substrates. In contrast, the 1-3 mismatched substrate produced less than the amount of recombinant (lane 3, Fig. 4).


Figure 4: Effect of mismatch location on strand joining in half-sites. A, the substrates for the joining reactions are schematically represented. The 5` end-labeled strand was linked at the 3`-phosphate to a short Flp peptide via the active site tyrosine (see ``Materials and Methods''). Phosphoryl transfer to the 5`-hydroxyl group of the acceptor strand (thick line) was assayed. Each substrate harbored three nucleotide mismatches between the bottom strand and the phosphoryl acceptor strand. Their locations are shown by the bubbles. B, the results of the assay with each of the substrates in A are displayed. The substrate and strand transfer product bands are represented by S and P, respectively.



The results obtained from the coupled strand cleavage/union assay (Fig. 3, C and D) and the strand union assay uncoupled from cleavage (Fig. 4) attest to the critical role played by base pairing in the joining reaction. The Watson-Crick complementarity at the strand joining end may anchor the 5`-hydroxyl in the proper alignment with the phosphotyrosyl bond, or base pairing may facilitate the Flp-DNA contacts required for accurately positioning the hydroxyl group.

Recombination between Full Sites and Half-sites: Effects of Spacer Nonhomologies

Previous results (Qian et al., 1992; Serre et al., 1992) and data not shown here demonstrate that in a cross between a half-site and a full site, the strand transfer product is derived predominantly from the alignment in which the half-site and full-site spacers are in the homologous configuration at spacer positions 1 and 2 (or 1` and 2`). There is a strong bias against the reverse alignment between the two substrates. It would appear that homology at the third spacer position (3 or 3`) is also sensed in this discrimination, although this position is less critical than the other two (data not shown). The requirement for homologous pairing during strand joining reactions in half-sites (see Fig. 4) provides a rational explanation for the observed bias in productive alignment during half-site to full-site reactions. The half-site data also indicate that, for a strand joining event, homology beyond the three adjacent positions between partners (equivalent to base complementarity within a substrate) would be irrelevant. To address this issue, we constructed two sets of three half-sites each (Fig. 5). One set had the 5`-TT-3`/3`-AAA-5` configuration (positions 1 and 2/1-3); the other had the 5`-TC-3`/3`-AGA-5` configuration (positions 1 and 2/1-3). Within a set, the five remaining spacer positions on the exchange strand were designed to be complementary to the top strand spacer positions of the full site or to the bottom strand spacer of the full site or to have no complementarity with the top or the bottom strand spacer. The P-labeled half-sites were reacted with the normal unlabeled full site (Fig. 5). The strand transfer product obtained by the left and right alignment of a half-site with respect to the full site partner (see Fig. 1) are called FPL and FPR, respectively, in Fig. 5. The recombinant products from the TT/AAA class of half-sites showed the same large bias toward the left alignment regardless of the spacer sequence at positions beyond the first three (4, 4`, 1`-3`). Conversely, the TC/AGA class of half-sites showed a similar bias toward the right alignment. Thus, the three spacer positions next to the cleavage site, in each case, swayed the outcome in their favor even when overall spacer homology would have suggested the opposite outcome.


Figure 5: Strand transfer between a half-site and a full site. The two sets of half-sites and the full site used in the crosses are shown. Reactions with the TT/AAA set of half-sites are presented in A; those with the TC/AGA set are shown in B. The half-sites were phosphorylated at the 5` end of the cleavage strands with P (*) and at the 5` spacer terminus of the noncleavage strand with unlabeled phosphate. Phosphorylation of the spacer hydroxyl prevented recombination within or between half-sites. All reactions contained the unlabeled full site. The lane numbers corresponding to reactions containing a particular half-site are shown against its noncleavage strand. The products of strand transfer via left and right alignments are marked FPL and FPR, respectively. S denotes the labeled substrate band.



Recombination between Full Sites Harboring Spacer Nonhomology

How does the influence of spacer homology (seen in the half-site and half-site versus full-site reactions) play out in full-site versus full-site reactions? To answer this question, we built three full-site substrates (see Fig. 6). In these substrates, base complementarity at a spacer position is represented by two identical symbols, one filled and the other unfilled. A mismatched position is shown by a pair of non-identical symbols. One of these substrates, S1, has a perfectly matched spacer; the other two, S2 and S3, have a left end bubble (mismatches at positions 1 and 2) in their spacers. In the presence of Flp, the mismatched full sites (S2 and S3) produced higher levels of the cleavage product than the matched one (S1) (compare the cleavage bands, CL, in lanes 2, 5, and 8, Fig. 6). This disparity is consistent with the decreased ability of the mismatched substrate to reseal the nick either within itself or by strand transfer to a partner. Three full-site crosses are represented in Fig. 6. In each cross an end-labeled full site (S) was reacted with an unlabeled concatenated partner (Sn; average length of 3 monomeric units) obtained by a normal ligation reaction (see ``Materials and Methods''). We had noted that the length of the full-site substrates influences the efficiency of strand transfer (the longer substrates are better reactants). When labeled S2 was crossed with the unlabeled S3 oligomer (S3n), both strand cleavage and strand transfer products (CL and P, respectively) were readily formed (lane 6, Fig. 6B). In the absence of S3n, only the cleavage band was produced as expected (lane 5, Fig. 6B). Recall that S2 and S3 are each mismatched at the left end; yet, when the cleaved strands switch partners, a perfect match would ensue. When labeled S3 was crossed with unlabeled S3n, recombinants were not detected (lane 9, Fig. 6C). However, a prominent strand cleavage band was seen in the presence and absence of S3n (lanes 8 and 9, Fig. 6C). The intensity of the cleavage band in an S3 times S3n reaction was roughly equivalent to the sum of the intensities of the cleavage and recombinant bands in the corresponding S2 times S3n reaction. We conclude that S3 is cleaved as well as S2, yet nonhomology precludes strand joining between two S3 molecules. Similar results were obtained with the S2 versus S2n reaction as well (data not shown). The fully matched cross between labeled S1 and unlabeled S1n yielded the cleavage product and the recombinants as expected (lane 3, Fig. 6A).


Figure 6: Effect of spacer heterology on recombination in full-site substrates. Reactions were carried out with an indicated full site (S1, S2, or S3) labeled on the top strand at the 5` end (*) and an unlabeled, concatenated full-site partner (S1n or S3n). Position 3 within the spacer is T/A for all of the substrates. Other spacer positions are symbolically represented. Complementarity at a position is indicated by a pair of identical filled and unfilled symbols. For example, in the reaction shown in B, the substrates carry mismatches at spacer positions 1 and 2 (open diamonds and closed circles in S2; open circles and closed diamonds in S3n). Strand swapping between them produces perfect matches at these positions. The bands corresponding to substrate, strand transfer product, and cleavage product are indicated by S, P, and CL, respectively. The heterogeneity of CL arises from incomplete digestion by proteinase K of the Flp moiety linked to the 3`-phosphate of the cleavage product.



In experiments not detailed here, we also tried to mimic the attP homoduplex X attB heteroduplex reaction in which only one attB strand is homologous to attP (Bauer et al., 1985). The assays were done by crossing two end-labeled heteroduplex (mismatched at spacer positions 1 and 2) full sites with an unlabeled homoduplex full site in separate reactions. The mismatches were designed such that crossover following cleavage would produce a cohesive or a noncohesive end, respectively, for union with the labeled strand of the two half-sites. The kinetics of cohesive end joining followed that of strand joining in a normal cross between two homoduplex full sites. The noncohesive end joining reaction displayed markedly slower kinetics (data not shown). The mismatch-induced kinetic delay paralleled that observed in the half-site joining reaction (see Fig. 2, C and D).

Effect of Nonhomology on Strand Transfer: Decreased Joining or Increased Reversal of Joining?

The failure to detect recombinants in the S3 times S3n reaction (Fig. 6) could either be due to a true joining defect or due to reversal of the joining reaction by an increased rate of cleavage when the spacer positions 1 and 2 are in the mismatched configuration. To test these possibilities, cleavage reaction was assayed in six synthetic substrates schematically shown in Fig. 7. They mimic joined products resulting from strand union between full-sites heterologous or homologous at spacer positions 1 and 2. The four-armed substrates (Fig. 7, E and F) are the closest mimics of the Holliday intermediate formed by a pair of reciprocal exchanges during a normal recombination event. Synthetic Holliday junctions analogous to the ones used here have been shown to be resolved by Flp into expected products (Jayaram et al., 1988; Dixon and Sadowski, 1993). All substrates were assembled by hybridization of appropriate oligodeoxynucleotides and were labeled at the 3` end on one of the component strands to monitor strand cleavage.


Figure 7: Strand cleavage within substrates that mimic strand union products harboring spacer mismatches. The substrates (linear molecules or Holliday junctions) used in the assay were radioactively labeled at the 3` end (*) on the indicated strand. Spacer mismatches are indicated by bubbles. There was no spacer mismatch or spacer nick in the control substrate in reaction A. Substrates in reactions B and E harbored mismatches at positions 4 and 4`; those in reactions C,D, and F contained mismatches at positions 1 and 2. Substrates in B, C, E, and F carried a medial nick within the spacer. The time points at which cleavage was assayed in the linear substrates are indicated under the lanes. Cleavage in Holliday junctions (E and F) was monitored at 2.5 min in reactions containing approximately 0.01 pmol of the labeled substrate. The molar ratios of Flp to Flp binding element in these assays were 0, 0.25, 0.5, 1, and 2 (from left to right). S and CL stand for substrate and cleavage bands, respectively.



Cleavage on the labeled top strand would dissociate the linear substrates shown in Fig. 7, B and C (containing a medial nick in the spacer) into two half-sites as the 3-bp hydrogen bonding between them would not be stable enough at 30 °C (at which temperature the assays were done). The design of these full sites also ensured that spacer positions 1-3 and 1`-3` on the labeled strand were mismatched. This was to discourage a top strand cleavage event from being converted to a hairpin product following bottom strand cleavage. Thus the labeled product from virtually all cleavage events would be trapped. The kinetics of strand cleavage in these two substrates was roughly equal (CL bands in Fig. 7, B and C) and comparable with that of the substrate in Fig. 7D that is mismatched at spacer positions 1 and 2, but contains no spacer nick. Essentially all cleavage events in the reaction (Fig. 7D) would also be trapped because strand joining would be slowed down by the spacer noncomplementarity. There was little accumulation of the cleavage product from the substrate that harbored no spacer mismatches and no medial nick (Fig. 7A). In this reaction, nicks resulting from cleavage could be efficiently resealed by strand joining. Faint cleavage bands could be detected in Fig. 7A upon longer exposures of the autoradiogram. The Holliday junction in Fig. 7F (mismatches at positions 1 and 2) did not reveal enhanced cleavage relative to its counterpart in Fig. 7E (mismatches at positions 4 and 4`) as judged by the release of the short labeled oligodeoxynucleotide product (compare CL in Fig. 7, E and F). In fact, slightly elevated cleavage (1.5-2-fold) was noticed when the noncomplementarity was at 4 and 4` rather than at 1 and 2.

From a measure of cleavage events trapped by dissociation of duplex substrates into two halves (Fig. 7, B and C), or as a result of spacer mismatches at the joining end (Fig. 7D) or by the release of the cleavage product from the Holliday junctions (Fig. 7, E and F), we conclude that the reversal of the joining reaction is not affected significantly by spacer mismatches at positions 1 and 2. The failure to detect strand transfer products in reactions of full sites harboring these mismatches (Fig. 6C) must therefore reflect an authentic defect in the strand joining step.

Thus the effects of spacer mismatches on the strand union step in half-site reactions and of spacer nonhomology in the half-site to full-site reactions can be faithfully reproduced in full site times full-site reactions.

Strand Joining in Full Sites Is Insensitive to Nonhomology beyond the Three Positions Adjacent to the Reaction Center

Half-site reactions and half-site to full-site reactions demonstrated that base complementarity beyond the three spacer positions adjacent to the scissile phosphodiester has little effect on the strand joining reaction. Does this rule hold in the reaction between two full sites as well? Four reactions schematically represented by B-E (Fig. 8) compared with the control reaction A provided the answer. Each member of the S series of substrates was end-labeled on the top strand at the 5` end and crossed with the indicated partner from the unlabeled, concatenated Sn series. All substrates contained spacer mismatches at positions 1 and 2. These mismatches were so designed as to remain unmatched in both partners following strand swapping (C) or to be matched in only one partner (D, E), or to produce matches in both partners (A, B). The third position in the spacer was homologous in all substrates (T/A). The remaining five spacer positions (4 and 1`-4`) were either completely homologous (A) or completely nonhomologous (B-E) between two partners of a cross.


Figure 8: Position effects of heterologies on strand transfer between two full sites. The general design of substrates and strand transfer reactions are as described for Fig. 6. The labeled substrate S2 (A) or S4 (B-E) was crossed with the indicated unlabeled concatemer (S2n, S3n, S5n, or S6n). At a particular spacer position complementarity is indicated if the top and bottom strands carry the same symbol, one filled and the other unfilled. Two non-identical symbols at a position indicate a mismatch. The five spacer positions distal to the top strand cleavage point (4 and 4` to 1`) are homologous between partner substrates for the reaction in A and heterologous for the reactions in B-E. All substrates contain the same base pair T/A at spacer position 3. The end-labeled substrate is marked by the asterisk at the 5` terminus of the top strand. The substrate, cleavage product, and strand transfer products are denoted by S, CL, and P, respectively.



The reactions between S and Sn represented by A and B would produce perfect matches at positions 1-3 following strand swapping. However at spacer positions 4 and 1`-4`, the reaction partners were completely homologous in the A reaction and completely heterologous in the B reaction. Yet the yields of the recombinants (P) from the two reactions were nearly identical (compare lane 3 of A to lane 6 of B, Fig. 8). The S and Sn substrates in C harbored equivalent mismatches at positions 1 and 2 and contained the same heterologies at 4, 1`-4` as in B; however, unlike B, they could produce no reciprocal match following strand swapping. No recombinants were obtained in reaction C (lane 9 of C, Fig. 8), although strand cleavage was comparable with that in B (compare CL in lane 6 of B and lane 9 of C, Fig. 8). In the D and E reactions, the heterologies at 4, 1`-4` were identical between the S and Sn partners. The 1 and 2 mismatches were such that in D strand swapping would produce a match within S4 (the labeled substrate) but not within S5n (the unlabeled substrate). The reaction resulted in labeled recombinants (lane 12 of D, Fig. 8). Strand swapping in E would produce a match within S6n, but not within the labeled S4. No labeled recombinants were detectable in this reaction (lane 15 of E, Fig. 8). When S4 was labeled at the 3` end rather than at the 5` end, recombinants were indeed observed as predicted (data not shown). In the absence of the oligomeric partner, the labeled substrates yielded the cleavage product (CL) in reactions A-E (lanes 2, 5, 8, 11, and 14, Fig. 8).

These results demonstrate that the strand joining reaction in full sites is insensitive to nonhomology beyond the three spacer positions adjacent to the scissile phosphodiester, exactly as predicted from half-site reactions and half-site times full-site reactions.


DISCUSSION

We have addressed here the role of spacer homology in Flp recombination using half-sites and full-site substrates containing appropriately positioned mismatches or substrate pairs harboring suitably placed heterologies. We come to the surprising conclusion that homology is tested prior to the formation of a covalently joined Holliday junction. A critical homology-sensitive step is the strand joining reaction itself. The results fit into a model in which branch migration of the Holliday intermediate can be limited to the very central region of the spacer.

Base Complementarity as a Mechanism for Orienting Reactive Groups

The experimental results presented here demonstrate a direct and clear-cut effect of Watson-Crick complementarity on the strand joining step of the Flp reaction. In half-sites and in full sites, the strand joining event requires base pairing at the three positions adjacent to the 5`-hydroxyl group carrying out the nucleophilic attack. The strongest contributions to pairing are made by the two neighboring bases nearest to the hydroxyl group. When these cannot be base paired with the partner strand, the joining reaction is strongly depressed. The effect of noncomplementarity at the third position on the joining reaction is less pronounced. Thus it is attractive to imagine that the Flp recombinase utilizes the base pairing potential within DNA to physically anchor the hydroxyl group in its attacking position. One cannot rule out the possibility that base pairing plays an indirect role in facilitating a critical protein-DNA contact that then orients the 5`-hydroxyl group. However, the basic simplicity of the base pairing mechanism to accomplish directed placement of a reactive species is quite appealing.

That the physicochemical attributes of the nucleic acid structure are tapped by the recombinase enzyme to facilitate phosphoryl transfer is not particularly surprising. A simple parallel may be drawn between the joining reaction mediated by Flp and the DNA ligation reaction carried out by Escherichia coli DNA ligase (Lehman, 1976). The nucleophilic attack by the 3`-hydroxyl on the 5`-phosphate activated by adenylation during ligase action requires cohesive DNA ends under normal reaction conditions. In the Flp and ligase examples, the hydrogen bonding potential within the substrate is exploited by the enzyme to stabilize a structure that is conducive to the chemistry that it performs. A more direct role for a DNA substrate in catalysis has come to light in the cleavage reaction by EcoRI and EcoRV restriction enzymes (Jeltsch et al., 1993). Here, activation of the attacking water molecule is accomplished by the pro-Rp nonbridging oxygen of the phosphate group that is the immediate 3` neighbor of the scissile phosphodiester bond. In the extreme example of self-splicing RNAs, the distinction between substrate and catalyst virtually breaks down. In the case of the Tetrahymena ribozyme, the structural and functional attributes of the active site are derived from specific Watson-Crick and non-Watson-C