(Received for publication, January 13, 1995; and in revised form, June 15, 1995)
From the
Absolute homology between partner substrates within the strand exchange region (spacer) is an essential requirement for recombination mediated by the yeast site-specific recombinase Flp. Recent experiments suggest that 3-base pair homology adjacent to the points of exchange at each end of the spacer is utilized in a base complementarity-dependent strand joining reaction. Homology of the central 2 base pairs of the spacer is also critical, but how homology is tested at these two positions is unknown. We have addressed the role of homology-dependent branch migration in Flp recombination by assaying strand cleavage and resolution in a set of synthetic Holliday junctions in which the branch point is freely or partially mobile through the spacer, or is immobilized at each position within the spacer or immediately flanking it. A strong bias in the direction of Holliday resolution is observed only when the branch point is located just outside the spacer (at the junction of the Flp binding element and the spacer). A significantly smaller bias is noticed when the branch point is frozen immediately adjacent to this position within the spacer. Resolution in these cases is most often mediated by exchange of the scissile phosphodiesters at the branch point or proximal to it, and rarely by exchange of the scissile phosphodiesters distal to it. In light of these and previous results, we discuss possible checkpoints for testing partner compatibility during Flp recombination.
Recombination catalyzed by site-specific recombinases of the Int
family, for example, the Int protein, the Flp protein from Saccharomyces cerevisiae, and the Cre protein from phage P1,
proceeds in two steps of single strand exchanges, a Holliday junction
being an obligatory intermediate. The reaction is dependent on absolute
homology between partner substrates within the region of strand
exchange (or overlap segment, or spacer) (Craig, 1988; Sadowski, 1993).
The
Int reaction has been extensively explored with regard to its
requirement for homology between recombination partners. Results from a
large number of experiments (Weisberg et al., 1983; Bauer et al., 1984, 1985; Kitts and Nash, 1987; Nunes-Duby et
al., 1987, 1989; Cowart et al., 1991) can be accommodated
by a model in which branch migration of the Holliday junction across
the overlap segment precedes its resolution; non-homology 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, 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 classified as
a member of the Int family. Recent studies using half-site and
full-site substrates have yielded new insights into how homology is
utilized by the Flp recombinase in testing compatibility between
partner substrates (Lee and Jayaram, 1995). The first critical
functional role of homology during recombination appears to be in
aligning the 5`-hydroxyl group of a nicked DNA strand using
Watson-Crick base pairing during the strand joining reaction. The
results are consistent with a model in which homology-dependent branch
migration of the Holliday intermediate may be limited essentially to
the central 2-4 bp ()region of the spacer sequence
(see Fig.1). One implication of this model is that the
cross-over point might be fixed within the spacer during the strand
joining events that generate the covalently closed Holliday
intermediate and the strand cleavage events that lead to its
resolution.
Figure 1: A model depicting the homology-sensitive steps in one round of Flp recombination. According to the model, homology at the three cleavage-proximal spacer positions (123 for a reaction initiated a the left) is tested at the strand joining step following the first pair of cleavages. Homology at positions 4 and 4` is utilized for advancing the position of the cross, while concurrently isomerizing the reaction complex into the Holliday resolution mode. The location of the cross is between 3 and 4 during formation of the Holliday intermediate; the cross is placed between 4` and 3` immediately prior to the resolution step. For a reaction initiated at the right, the initial homology test occurs at spacer positions 1`2`3`, the branch point progresses through 4` and 4 and localizes itself between 4 and 3 in preparation for resolution. The model is borrowed from Lee and Jayaram(1995).
Synthetic Holliday junctions have been used as
substrates in the analysis of recombination reactions carried out by at
least three recombinases of the Int family: Int, Flp, and the
XerC/XerD proteins of Escherichia coli (Hsu and Landy, 1984;
Jayaram et al., 1988; Dixon and Sadowski, 1993; Kho and Landy,
1994; Arciszewaska and Sherratt, 1995). In principle, a Holliday
junction may be resolved in one of two modes, giving rise to
``parental'' or ``recombinant'' duplexes. In this
study, we have used a series of synthetic Flp Holliday junctions to
address the following question: does the placement of the cross-over
point within the spacer region constrain the resolution of the Holliday
junction to parentals or recombinants? The model proposed by Lee and
Jayaram(1995) supposes that some branch migration within the spacer may
be required (or occurs commensurate with) the switching of the
recombination complex from the Holliday forming mode (first pair of
exchanges) to the recombinant yielding mode (second pair of exchanges).
Arresting branch migration at specific points may then be expected to
introduce detectable bias in the direction of Holliday resolution. We
tested this notion by assaying the resolution of synthetic immobilized
Holliday junctions by Flp. While this study was in progress, Dixon and
Sadowski (1994) reported that Holliday structures in which the branch
point within the spacer was placed adjacent to one pair of Flp binding
elements or 4 or 5 bp away from them could be resolved by Flp. However,
a large bias in the direction of resolution was observed only with the
first of these three junctions. The Dixon-Sadowski results provided
further impetus to complete the analyses presented in this paper.
The 3` end
of a deoxyoligonucleotide was labeled with
3--
P-labeled cordycepin triphosphate using terminal
deoxynucleotidyl transferase enzyme. The excess unreacted cordycepin
triphosphate was removed by spin-dialysis on a Sephadex G-25 column.
Hybridization to the partner oligodeoxynucleotide(s) was done in TE.
A recent model for Flp site-specific recombination (Lee and
Jayaram(1995); see Fig.1) proposes that, following cleavage of
the two phosphodiester bonds at one end of the spacer (say left), three
nucleotides adjacent to the cut are swapped between partner substrates (Fig.1, A and B). After testing
complementarity between the swapped segments and the uncut strands,
ends are sealed to form a covalently closed Holliday intermediate (Fig.1C). At this stage of the reaction, the
cross-strand junction is placed 3 bp away from the point of initial
exchange (between spacer positions 3 and 4). An
isomerization of the recombination complex is thought to occur by which
the cross is positioned 3 bp away from the right end of the spacer
(between positions 4` and 3`; Fig.1D). This step would likely involve branch
migration through the central 2 bp of the spacer (4 and 4`). Cleavage of the phosphodiesters at the right end, three
nucleotide swapping (E) and strand joining (F) will
then yield reciprocally recombinant products. This model has striking
parallels to that arrived at for Int recombination from the
pattern of resolution by Int of Holliday structures in which the
junction is immobilized at different points within the spacer sequence
(Nunes-Duby et al., 1995). The relative efficiency of
resolution as well as the bias in the direction of resolution (exchange
of top strands or bottom strands) is found to be exquisitely dependent
upon where, within the spacer, the junction is frozen. These results
would be consistent with features of the Lee-Jayaram model for Flp
recombination outlined above. The issue then is: does the branch point
within the Holliday structure exert positional effects in the
efficiency and direction of resolution reactions mediated by Flp?
Figure 2: Resolution by Flp of synthetic Holliday structures in which the cross is mobile throughout the spacer or is confined to three central positions (between 3 and 3`). The two synthetic Holliday structures used in the assays are schematically represented. Each pair of horizontal Flp arrows stands for a wild-type Flp binding element (5`-GAAGTTCCTATAC-3`/3`-CTTCAAGGATATG-5`). The spacer sequences are spelt out (1 through 1`). Additional sequences present at the left and right borders of the Flp binding elements are not shown. The heterologous base pairs used to trap the branch point are shown in bold italics. The cleavage positions are indicated by C1 through C4 (on strands 1 through 4). The arrows emanating from the cross-point span the range of branch migration possible within a junction. Following the resolution assay (see ``Experimental Procedures'' for details), the cleavage and strand transfer products were fractionated by electrophoresis in denaturing polyacrylamide and identified by autoradiography. For each substrate, the results from two separate assays are displayed. In each assay, only one strand was labeled at its 3` end (indicated by the asterisk). The substrate band is represented by S and the cleavage band by C. The resolution products are denoted by R. The radioactively labeled resolution products derived from cleavage at C1 and C2 are R1,3 and R2,4, respectively. The predicted sizes of the cleavage bands C1 and C2 are 35 and 38 nt, respectively. The corresponding resolution products would be 64 nt (R1,3) and 70 nt (R2,4).
Figure 3: Synthetic Holliday structures in which the junction is trapped at each position within the spacer or immediately adjacent to it. The schematic representation of the Holliday structures generally follows the same rules as those outlined in Fig.2. The sizes of the cleavage and resolution products are also the same as in Fig.2. In two of the structures (A and I), the branch point is immobilized between the last base pair of the Flp binding element (position 0 or 0`) and the first bp of the spacer (1 or 1`). To introduce heterology at the spacer-proximal position of the binding element, the normal T-A bp was altered to C-G (shown in bold uppercase letters). This alteration does not affect Flp binding as inferred from the ability of the variant target site to undergo recombination with approximately wild type efficiency (Senecoff et al., 1988; Cox, 1988).
Figure 4: Resolution of immobilized junctions by Flp. Results from two separate assays with each Holliday structure (A-I, see Fig.3) carried out as described under Fig. 2are shown. The symbols S, C, and R refer to substrate, cleavage product, and resolution product, respectively. The asterisk stands for the 3` end-label.
The resolution bias observed for the various junction substrates could result from a bias in strand cleavage, or in strand joining following cleavage, or may reflect the balance between these two reactions. Lee and Jayaram(1995) observed that the strand joining reaction uses base complementarity primarily at the two spacer positions adjacent to the nick, with a minor contribution from the third position as well. Spacer positions beyond the third nucleotide appear to have little effect on the kinetics of joining. In the case of the left end junctions A and B, there is no heterology at the right end and hence no bias in joining imposed by base non-complementarity. On the other hand, at the left end, heterology would favor joining in the parental mode rather than in the resolution mode. As a result, the bias toward left resolution observed here for A and B is likely to be underestimated. By similar arguments, the right bias for H and I would also be under-represented. As the heterology responsible for trapping the cross is moved away from spacer positions that influence the joining reaction, interference to resolution by non-complementarity should fade. The relatively higher levels of resolution products obtained with such junctions (for example, compare D, E, and F to A and B, and H and I in Fig.4) agree with this idea.
The above results complement and extend those obtained by Dixon and Sadowski(1994). We find that the lack of resolution bias they observed at two positions within the spacer holds true for several other positions internal to the spacer. In addition, the strong resolution bias seen at the exchange point flanking the spacer at one end is reproducible at the other end as well, but in the opposite direction.
The Flp site-specific recombination is exquisitely sensitive
to spacer non-homology between DNA partners at every nucleotide
position within the 8-bp strand exchange region. A single base pair
spacer heterology between partner substrates is sufficient to reduce
recombination to undetectable levels under in vitro reaction
conditions (Andrews et al., 1986; Senecoff and Cox, 1986;
Dixon and Sadowski, 1994). According to a recent proposal (Lee and
Jayaram, 1995; see Fig.1), part of the spacer homology is
utilized in ensuring strand joining between correct partner substrates.
The model speculates that the remainder of the homology is likely
involved in coupling limited branch migration of the Holliday
intermediate to an isomerization step in which the active site
configuration of the recombinase tetramer is reorganized to perform the
resolution reaction. Observations regarding resolution of synthetic
Holliday junctions by the Int protein (S. Nunes-Duby et
al., 1995) fit into a model that has close parallels to features
of the Lee-Jayaram model. They found that a Holliday junction in which
the branch can migrate through just the central base pair of the
overlap region, but is blocked 3 bp from the exchange points at either
end, is resolved by Int with roughly equal probability to parentals or
to recombinants. Furthermore, in two junctions in which the strand
crossing is trapped between the central base pair and its immediate
left neighbor or its immediate right neighbor, the resolution reactions
show a large bias, toward parentals in one case and toward recombinants
in the other. It would seem likely therefore that the limited
requirement for branch migration in the Flp and Int systems might serve
the same functional purpose.
Impressed by the apparent congruence of the roles of homology in Int and Flp recombinations, arrived at from two widely different approaches, we tested whether the correlation between the location of the cross and the mode of Holliday resolution seen with Int is reproducible with Flp as well. We find that a fully mobile junction, or one restricted in mobility through the central 2-bp core of the spacer, is resolved efficiently by Flp with no obvious bias in the direction of resolution. This result is analogous to that obtained with Int and is fully expected from the Lee-Jayaram model. However, our results and those of Dixon and Sadowski(1994) show a general lack of large resolution bias internal to the spacer, regardless of the spacer position at which a junction is locked in. In this regard, the Flp and Int results are quite distinct. Only when the junction is immobilized at the normal exchange points to the immediate left or the immediate right of the spacer does a strong preference in the resolution mode become apparent with Flp. In summary, the inference from the Lee-Jayaram model that an immobilized Holliday structure resolved by Flp with a large bias in one direction must have a symmetric counterpart resolved in the opposite direction is satisfied by the experimental results recorded here. Intuitively, one would have expected these immobilization points to be 3 bp away from one spacer end in one case, and 3 bp away from the opposite spacer end in the other. In reality, they are located at either end of the spacer and not internal to it.
One interpretation of the results cited above is that the effect of spacer heterology in Flp recombination is much less pronounced than is generally believed. Provided a Holliday junction is formed with its branch point located away from the Flp binding element-spacer junction, it has roughly equal chances for being resolved into recombinants or into parentals. The failure then to obtain recombinants in presence of spacer heterology likely results from a reverse recombination of the spacer mismatched products before they dissociate from the reaction complex.
Can we envisage an
alternate reaction scheme for Flp recombination in which our present
results on Holliday junction resolution can be reconciled with the
basic features of the Lee-Jayaram proposal? In attempting to do so, we
rely primarily on the special architectural design of the Flp active
site (Chen et al., 1992, 1993; Yang and Jayaram, 1994).
Assembly of a functional strand cleavage pocket within a Flp monomer
bound adjacent to a scissile phosphodiester requires that the active
nucleophile (Tyr) be delivered in trans from a partner
Flp monomer bound across the spacer (Lee and Jayaram, 1994). This
delivery is accommodated by the large protein induced bend within the
spacer DNA (Schwartz and Sadowski, 1990; Chen et al., 1992b).
Similarly, the strand joining pocket within the same Flp monomer is
rendered functional when the 5`-hydroxyl group from the Flp-nicked
strand is delivered to it in trans and oriented within it via
Watson-Crick base pairing (Lee and Jayaram, 1995). Thus a Flp monomer
is able to perform a concerted cleavage-joining event through a
composite active site which recruits the appropriate reactive group for
each reaction from a second Flp monomer or from the Flp-cleaved DNA
substrate. In principle, therefore, two monomers of Flp separated by
the spacer can cooperate to assemble two active sites, a Holliday
forming pocket and a Holliday resolving pocket (Chen et al.,
1993). However, strong circumstantial evidence indicates that at one
time only one of the two active sites can be organized (Qian et
al., 1990). (
)For example, within a full-site occupied
by two Flp monomers, cleavages are almost exclusively single-stranded.
Similarly, within the dimeric form of a Flp-half-site complex, the
maximum obtainable cleavage corresponds to cutting at half of the
scissile phosphodiesters. Exclusion of the second active site upon
assembly of the first is functionally analogous to the phenomenon of
half of the sites activity seen in certain allosteric enzymes. Thus,
for successful completion of recombination, the active sites mediating
formation of Holliday intermediate (at one end of the spacer) must be
disassembled, and those mediating its resolution (at the other end of
the spacer) must be assembled within the two ``across the
spacer'' Flp pairs. (
)We consider this
disassembly-assembly process to be synonymous with the branch migration
(or isomerization) step that is central to the Lee-Jayaram model (see Fig.1).
Given the shared active site and half of the sites
activity of Flp, one may imagine that, during one round of
recombination, the reactive proteinDNA complex can exist in one
of two metastable structural forms (Fig.5; form III and its
symmetric counterpart). These would correspond to locking in of active
site configuration 1 or active site configuration 2, and result
directly from the nature Flp-Flp interactions across the spacer. One
would expect that in these structures the branch point is likely to be
constrained. The Holliday resolution experiments support this notion.
They indicate that the points of restraint are located at the left or
right borders of the spacer region. According to Lee and Jayaram(1995)
partner compatibility between DNA substrates is not tested till after
strand cleavage has occurred. If nonhomology exists at the three spacer
positions adjacent to the cleaved end, formation of a covalent Holliday
intermediate is inhibited. The active site configuration 1 will not be
disengaged, and the preferred joining reaction will be the reversal of
cleavage. Hence parental configuration of the DNA partners will be
restored. If these spacer positions are homologous, formation of
Holliday intermediate will occur. Under normal conditions (no
heterology within the spacer), isomerization of the recombination
complex drives the branch point to the position at which active site
configuration 2 is engaged and resolution proceeds in the recombinant
mode. According to the Lee-Jayaram model, this transition point could
potentially be located 3 bp away from the resolution point or at the
resolution point itself. Lee and Jayaram preferred the former case
because of the limited branch migration involved and because of the
symmetry conferred by this arrangement on the first and second pair of
cleavage-joining reactions. If heterology beyond the three spacer
positions adjoining the initial cleavage point precludes active site
configuration 2 or inhibits strand joining in the recombinant mode
following cleavage by active site 2, the recombination complex reverts
to active site configuration 1 whose structural constraints force the
cross-over point to be trapped at the spacer extremity (form III, Fig.5). Hence resolution can occur only in the parental mode,
and no recombinants can be produced. Thus, regardless of whether the
reaction is initiated at the left or at the right, effect of spacer
heterology would be to freeze Holliday junctions at the initiation end.
In either situation, recombination would be aborted by resolution in
the parental mode.
Figure 5:
Potential configurations of the Flp
tetramerDNA complex during recombination. In accordance with the
model shown in Fig.1, the normal reaction involves two
transient Holliday structures, form I and form II. Transition from I to
II is associated with a reversal of catalytic roles between the two Flp
monomers at the left and those at the right (switch from active site
configuration 1 to active site configuration 2). The distinct
contributions by each pair of Flp monomers to the reaction (orientation
of the phosphate or donation of Tyr
) are indicated by the hatched or unhatched ovals. For a reaction initiated
at the left, blocking the conversion of form I to form II or of form II
to recombinants results in the metastable form III. In this state,
active site configuration 1 is established and the cross is trapped at
the left end of the spacer. A corresponding metastable form III` exists
for a reaction started at the right, in which active site configuration
2 prevails and the cross is trapped at the right spacer end. Resolution
within these structures can only give rise to the
parentals.
Our current results may now be explained as follows. This explanation must be tempered by the caveat that preformed Holliday structures are likely to permit greater degrees of freedom in protein-protein interactions upon Flp binding than are feasible in similar structures produced during the act of recombination. In all likelihood, reactions with these artificial substrates cannot exactly reproduce, but only approximate the natural situation. When Flp monomers associate with synthetic Holliday junctions immobilized at positions within the spacer, either active site configuration 1 or 2 can be established with roughly equal probability. As a result, resolution in the parental or recombinant mode is equally likely. However, during a regular recombination reaction (where the first exchange is mediated by Flp rather than the experimenter), such Holliday structures may be too short lived to contribute significantly to the reaction (even when their presence might be expected based on heterology). A stalling branch point, resulting from spacer heterology, is forced to its limit position by the nature of the metastable states accessible to the recombination complex, and is thus resolved in the non-recombinant mode. Only two of the synthetic junctions used in our experiments, those in which the cross-over point lies between 0 and 1 or 1` and 0`, would mimic the situation in which the active sites responsible for initiating the reaction (at the left or the right of the spacer) are frozen in that configuration. We find that these two junctions are resolved with a large bias, and in the expected direction. The smaller resolution bias observed with junctions trapped between 1 and 2 and 2` and 1` is most easily accommodated by a ``spill-over'' effect. We have no satisfactory explanation for the resolution preference (though small) obtained when the branch point is frozen between spacer positions 4` and 3`. We feel that this is a peculiarity of the particular Holliday substrate used rather than a special feature of the recombination reaction. Another Holliday structure immobilized at the same position was found to be resolved equally well in either direction by Dixon and Sadowski(1994).
Overall, the results summarized here on Holliday resolution together with the earlier findings of Lee and Jayaram(1995) indicate that partner discrimination during Flp recombination occurs at several levels. First, following cleavage the spacer segment is subjected to a limited homology test prior to strand joining and formation of a covalently closed Holliday junction. A second homology test is applied during the disengagement of the first pair of Flp active sites (responsible for Holliday formation) and assembly of the second pair of active sites (responsible for Holliday resolution). If heterology is detected at this step, disassembly is blocked, and the active site pair reverses the reaction to yield parental, and not recombinant, molecules. Assuming that the resolution pockets are assembled and that they proceed to execute strand cleavage, a third homology test comes into play at the joining step required to yield the mature recombinant products. If a fraction of molecules can escape each of these check points, total exclusion of recombination between two DNA substrates harboring spacer heterology will not be possible. However, if product dissociation from the recombination complex is a slow step, the system will have yet a final chance to eliminate the progeny of an illegitimate union by reversing the reaction.