(Received for publication, October 12, 1994; and in revised form, December 16, 1994)
From the
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.
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 ()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.
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
-
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.
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.
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.
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.
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.
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).
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 full-site reactions.
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 full-site reactions.
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.
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