(Received for publication, May 12, 1995)
From the
When two ongoing FLP-mediated recombination reactions are mixed, formation of cross-products is subject to a lag of several minutes, and the subsequent rate of cross-product formation is greatly reduced relative to normal reaction progress curves. The lag reflects the formation of a stable complex containing multiple FLP monomers and two FLP recombination target-containing DNA recombination products, a process completed within 5-10 min after addition of FLP recombinase to a reaction mixture. The reaction products are sequestered within this complex for an extended period of time, unavailable for further reaction. The length of the lag increases with increasing FLP protein concentration and is not affected by the introduction of unreacted (non FLP-bound) substrate. The results provide evidence that disassembly of FLP complexes from products occurs in a minimum of two steps. At least one FLP protein monomer is released from reaction complexes in a discrete step that leaves the reaction products sequestered. The recombination products are released in a form free to react with other FLP recombination target-containing DNA molecules only after at least one additional disassembly step. One or both of these disassembly steps are rate limiting for reaction turnover under conditions often used to monitor FLP-mediated recombination in vitro.
The FLP recombinase (M 48,794) is encoded
by the 2-micron plasmid of the yeast Saccharomyces cerevisiae,
and promotes a site-specific recombination reaction at sequences within
the same plasmid(1, 2) . FLP is a member of the
integrase family of recombinases, which includes the Cre recombinase of
bacteriophage P1 and the Int recombinase of bacteriophage
, among
others(3, 4) .
The site at which FLP recombinase
acts is called the FLP recombination target (FRT). ()The
minimal FRT consists of 2 inverted repeats of 13 base pairs, each
flanking an 8-base pair spacer. The 13-base pair repeats serve as FLP
protein binding sites; thus, there are two protomer binding sites per
minimal FRT or 4 per recombination reaction. The sequence of the wild
type FRT spacer is asymmetric, and alignment of two spacer sequences is
one of the factors that determines the course of an FLP-mediated
recombination reaction. If the spacer is replaced with a symmetric
(palindromic) sequence, two reacting FRTs can align in either of two
orientations, resulting in a new but predictable set of reaction
products. This feature of the symmetric spacer is useful for in
vitro analysis of FLP protein-catalyzed reactions.
The FLP reaction involves four DNA cleavage and rejoining reactions, occurring sequentially in pairs. A Holliday structure is formed after the first reciprocal set of cleavage and religation events. After an isomerization step, a new Holliday intermediate is resolved to products via the second set of cleavage and ligation steps. Much of the chemistry and an outline of reaction steps in these reactions has been elucidated(5) .
The interaction between FLP protomers in the
protein-DNA complex has recently come under close scrutiny. Each active
site for FLP-mediated DNA cleavage and strand exchange includes amino
acid residues contributed by two different FLP monomers. The
nucleophilic tyrosine involved in a given cleavage reaction is derived
from a monomer distinct from that bound on the adjacent 13-base pair
repeat (trans cleavage)(6, 7) . Schwartz and Sadowski (8, 9) have observed that FLP recombinase induces
sharp bends in its FRT substrate, which require strong protein-protein
interactions, and that this bending is required for FLP protein
catalysis. When FLP protein is incubated with FRT half-sites (FRTs that
have been cleaved in the spacer to generate a partial site with only
one FLP binding site), dimeric and trimeric protein complexes are
formed with bound DNA that are held together only by noncovalent
interactions and have a half-life of at least 1-2 h (10) . Recent experiments suggest that a pair of cleavage and
strand exchange reactions may be carried out by a complex containing
three tightly bound FLP monomers. ()
These strong protein-protein interactions led us to ask about the pathway for disassembly of an FLP-FRT complex. Since the disassembly step(s) should be the reverse of the assembly steps at the beginning of a conservative site-specific recombination reaction, examining disassembly might provide clues about assembly as well. There are several possible pathways for complex disassembly, and two are illustrated in Fig. 1. In the first pathway, FLP protein binds to an FRT; two protein-bound FRTs are brought together, react to form products, and then separate into two protein-DNA complexes. The separated complexes continue on to another recombination event with a new FLP-FRT partner. This model provides for multiple recombination events without complete dissociation of FLP protein. A second possibility would involve the release of FLP protomers from the protein-DNA complex after an FLP recombinase-catalyzed recombination reaction as a prerequisite to further reaction. In option one, the action of FLP protein is processive in that multiple recombination events may occur before the FLP protein dissociates from the FRT. There are many versions of option two; FLP dissociation could occur in a single step or as multiple steps of one or more monomers at several points in the reaction. Our experiments were designed to shed light on the disassembly pathway. We report here that a discrete step involving dissociation of one or more protein monomers from the FLP-product complex occurs prior to the step in which product FRTs are released in a form free to react with new FRT partners.
Figure 1: Potential dissociation pathways for the FLP-FRT protein-DNA complex. Two types of dissociation pathways are shown. In both cases, FLP protein binds its DNA target and catalyzes a reaction to products. On the left, these products dissociate from the reactive complex as protein-DNA complexes ready to react with other protein-DNA complexes. According to this model, protein dissociation from the FRT is not required for reactions with new FRT partners to occur. On the right, FLP protein must dissociate from the reactive complex to free the FRT products for new rounds of reactions. This dissociation may be concerted or may occur in a stepwise manner.
Figure 2: Absorbance spectra of native and denatured FLP protein. Spectra were obtained at 25 °C in FLP reaction buffer without polyethylene glycol (native) or the same buffer with 6 M guanidine HCl (denatured). The solutions yielding the spectra shown contained 774 nM FLP protein.
A total of eight determinations yielded an average extinction
coefficient of = 6.75
± 0.26
10
M
cm
or
= 1.4 A
mg
ml in FLP dilution
buffer at 25 °C.
Figure 3: Substrates and expected products for FLP-mediated site-specific recombination with the plasmid pJFS39. The plasmid pJFS39 is shown, and the sequence of the symmetrical FRT site is expanded at the top of the illustration. The FRT site is indicated by a solidband in the subsequent linear DNA molecule representations. Cleavage with EagI or StyI gives linear DNAs with identical sizes but with the FRT located at different positions relative to the ends of the DNA. Reaction of the EagI DNA alone gives products P1 and P4. Reaction of the StyI-cut DNA gives products of different sizes called P2 and P3. Reaction of the StyI-cut DNA with the EagI-cut DNA results in the formation of P1, P2, P3, and P4, along with four additional cross-products designated CP1, CP2, CP3, and CP4. The size of each product (given in parentheses) reflects the DNA arms flanking the FRT sites that are joined by the recombination reaction. bp, base pairs.
In a few cases, experiments were carried out to see if unbound DNA substrates could react with pre-formed complexes of FLP protein on another substrate. Reactions were carried out as described above, except that 2 aliquots of FLP protein were added to reaction a. After a 30-min incubation at 30 °C, reaction mixture b (without FLP protein) was added to reaction a, and product formation was monitored as described above. Control reaction c was carried out as for the standard experiments.
We generated two substrates for FLP protein by cutting the plasmid pJFS39 with either of two restriction enzymes, StyI or EagI (Fig. 3). The two linear DNA substrates generated are identical in length and both contain a single FRT. They differ only in the placement of the FRT relative to the ends of the DNA. The FRT in pJFS39 has a symmetric spacer sequence. Reactions involving one of the substrates can have two different outcomes. If the substrates are aligned so that the sequences flanking the FRT are parallel, the subsequent reaction will generate products that are indistinguishable from substrates. If the substrates are aligned so that sequences flanking the FRT are antiparallel, two distinct products will arise from a recombination reaction, one larger and one smaller than the substrate. Because the FRT is positioned differently in the two different substrates, the sizes of the products generated from each are distinct (P1 to P4). Since they contain identical FRT DNA sequences, the two substrates can also react with each other. The various possible alignments of the two substrates with each other give rise to four additional cross-products (CP1 to CP4). The substrates have been designed so that all of the possible products and cross-products are distinguishable on an agarose gel. The expected products of reactions involving one or both substrates are illustrated in Fig. 3.
We compared the rate of product formation in two sets of reactions. In the control reactions, both DNAs were present in one tube when FLP protein was added, and cross-product formation was monitored for 120 min. In the second set of reactions, the two DNAs were incubated separately with FLP protein for 30 min (enough time to reach apparent equilibrium in most cases) and then mixed together, and the formation of cross-products was monitored for 120 min. When product formation was compared for the two types of reactions, we found that all products in the control reaction began forming immediately, including cross-products. In contrast, cross-product formation after ongoing reactions were mixed exhibited a lag of several minutes. A representative result is shown in Fig. 4. The lag in cross-product formation indicated that the FRT substrates and products of the two ongoing reactions were sequestered and thus unable to react with each other.
Figure 4: A prereaction of individual substrate DNAs suppresses cross-product formation. Two reactions were set up as described in the text and in Fig. 3, containing 10.7 nM FRT-containing DNA molecules, and 120 nM FLP protein. Lanes labeled C are markers, with StyI-cut pJFS39 and EagI-cut pJFS39 in lanes1 and 2, respectively. Lane3 contains the reaction of StyI-cut pJFS39 DNA after 30 min, while lane4 shows the corresponding reaction with EagI-cut DNA. These reactions were mixed at the 30-min time point, and the subsequent reaction is shown in the lanes under ``Mix after 30 min'' with time points at 2, 5, 7, 10, 15, 30, 60, 90, and 120 min after mixing shown in lanes1-9, respectively. The lanes under Control show the progress of a reaction in which the StyI- and EagI-cut substrates are mixed prior to addition of FLP protein. Time points are 0, 2, 5, 7, 10, 15, 30, 60, 90, and 120 min after addition of FLP protein in lanes1-10, respectively. Products and cross-products are labeled as in Fig. 3. Bands labeled I above the product bands are primarily Holliday structures(11) . Small bands labeled AC reflect aberrant cleavage at the FRT site and appear in many reactions at relatively late times(11) .
The lag in cross-product formation seen after mixing of reactions a and b was not due to loss of FLP recombinase activity. The control reaction shows that a 30-min incubation of FLP recombinase prior to reaction does not result in a loss of activity. The rate of the reaction is essentially identical to a reaction initiated by FLP protein that had not been incubated at 30 °C. In some reactions (including that in Fig. 4), the FLP incubation prior to the control reaction c was carried out in FLP storage buffer. In others, the FLP recombinase was incubated under standard reaction conditions, and reaction c was initiated by addition of the DNA substrates. There was no significant difference in the FLP-mediated reaction observed with these varied protocols (data not shown).
Figure 5: Suppression of cross-product formation as a function of FLP protein concentration. Reactions were carried out as described in the text and the legend to Fig. 4. Cross-product formation is plotted as the CP1/S ratio. Reactions in which the EagI- and StyI-cut DNA substrate were prereacted separately for 30 min before mixing are shown with closedsymbols, with the 0 time in the plot reflecting the time of mixing. Reactions with opensymbols are controls with no prereaction. All reactions contained 10.7 nM FRT-containing DNA molecules. Symbols and numbers at the right of the figure identify the concentration of FLP protein, in nM, in each experiment.
Since FLP protein was in excess in all reactions, the simplest explanation of this result is that a longer lag in product formation is directly related to higher levels of free or nonspecifically bound FLP recombinase. Free protein would affect association to or dissociation from the protein-DNA complex by mass action. FLP protein concentration should not affect the rate of reaction steps within a protein-DNA complex that has a constant number of bound FLP protomers. The observed lag and its dependence on protein concentration is consistent with an inhibition of an FLP protein dissociation step.
Figure 6: Time required to sequester FRT-containing DNA. Reactions were carried out as described in Fig. 4and Fig. 5with 300 nM FLP protein and 10.7 nM DNA, except that the prereaction time was varied. Numbers attached to symbols at the right of the figure denote the prereaction time in minutes.
Recombination experiments were repeated as above, with the exception
that only one of the FRT substrate prereaction mixtures (a) included
FLP protein. After 30 min, a second reaction mixture (b) containing
unreacted FRT DNA was mixed with the first, and product formation was
monitored as before (Fig. 7). These reactions show the same lag
in cross-product formation as reactions where both FRTs were prereacted
with FLP protein. As seen in Fig. 7, the unbound FRT substrate
introduced with reaction mixture b does participate in FLP-mediated
recombination after mixing, yielding the products P2 and P3 efficiently
after an 8-10-min lag. This is presumably due to the presence of
excess FLP recombinase in reaction a. We attribute the lag in formation
of P2 and P3 to a nonspecific binding of the excess FLP recombinase to
DNA sequences remote from the FRT. Similar lags in product formation in
FLP-mediated recombination reactions are observed when FLP is
pre-incubated with nonspecific DNA. ()
Figure 7:
Effect of prereacting only one of two
FRT-containing DNA substrates. Reactions were carried out as described
in the text. Final FLP protein and DNA concentrations were 300 and 10.7
nM, respectively. Individual reactions are
-
and
-
, plots of CP1/S
and P2/S, respectively, for a single control reaction in which the EagI-cut and StyI-cut substrates were mixed prior to
addition of FLP protein (no prereaction);
-
, time
course (CP1/S) after mixing for a reaction in which both of the
FRT-containing DNA substrates were prereacted with FLP protein for 30
min;
-
, time course (CP1/S) after mixing for a reaction
in which the EagI-cut substrate was prereacted with FLP
protein for 30 min but the StyI-cut substrate was preincubated
without FLP protein;
-
, a plot of P2/S time course after
mixing for the reaction in which the EagI-cut substrate was
prereacted with FLP protein for 30 min but the StyI-cut
substrate was preincubated without FLP protein. Note that P2 is derived
from the StyI-cut DNA substrate, which is not prereacted with
FLP protein in this last reaction.
This experiment was done in two ways. In one case (plotted on the graph), reaction a contained 10.7 nM FRT sites and 600 nM FLP protein. This reaction was incubated for 30 min at 30 °C, at which point a second reaction mixture of equal volume (75 µl) containing the other FRT site at 10.7 nM but no FLP protein was mixed with it, and the subsequent reaction followed. In this reaction the FLP concentration was diluted by 50% upon mixing with reaction mixture b, while the FRT substrate concentration remained constant throughout the experiment. In the second version of this experiment, reaction a contained 300 nM FLP protein and 5.4 nM FRT substrate in 150 ml. A small aliquot of a concentrated solution of the second FRT was added to this reaction mix after 30 min, bringing the final FRT concentration up to 10.7 nM. In this case, the FRT concentration increased from 5.4 to 10.7 nM at the mixing step, while FLP protein concentration remained constant at 300 nM. Both reactions produced similar cross-product formation lags (data not shown).
FLP protein undergoes a slow dissociation from a stable FLP protein-FRT complex before reaction products are free to react with new FRT partners. The dissociation occurs in at least two steps (Fig. 8). A first step involves disassembly of part of the complex. This is shown as dissociation of one monomer in Fig. 8, although the actual number of monomers released may be different. The product of disassembly step 1 is a complex in which product DNAs are still held together, unavailable for new reactions. The existence of this step is based on the observation that higher FLP protein concentrations delay the release of products to react again. The illustrated step would be affected by mass action in a way that would affect the partitioning of intermediate generated by disassembly step 1. Under the conditions used here and in many other studies, the rate-limiting process for catalytic turnover in FLP-mediated recombination is one or both of the disassembly steps shown in Fig. 8.
Figure 8: Model for FLP protein dissociation from recombination products. Disassembly of the FLP-FRT complex is shown as occurring in two steps. Disassembly step 1 generates an intermediate in which at least one FLP monomer has departed, but the FRT sites are still sequestered and unavailable for further reaction. Disassembly step 2 generates free FRT sites in the sense that they can now undergo additional reactions. The partially populated intermediate between disassembly steps 1 and 2 is discussed at length in the text.
These conclusions are derived from several observations. When two ongoing FLP protein reactions are mixed, there is a lag in cross-product formation. The simplest explanation for the lag is that products in a reaction that is ongoing are sequestered, unable to react with new DNA partners. This idea is further supported by experiments which demonstrate that the degree to which cross-product formation is suppressed depends on how long the two separate FRTs are allowed to react before being mixed. This apparently reflects the formation of a stable FLP-FRT complex during the prereaction that occurs before mixing. The delay in cross-product formation also exhibits a mass action effect. Higher concentrations of FLP protein increase the length of the lag and decrease the rate of subsequent cross-product formation. Since FLP protein is in excess in all the reactions described here, only reaction steps that involve a change in the number of bound FLP protomers (i.e. a binding or dissociation step) in the reactive complex should be affected by changes in FLP protein concentration. Since we see a decrease in system turnover, we believe the delay in cross-product formation is due to an inhibited dissociation step. If the dissociation step producing the lag resulted in release of FRT reaction products to a substrate pool available for further reaction, no dependence of the lag in cross-product formation on protein concentration would be evident. Hence, the product of the dissociation step affected by protein concentration must be an FLP-FRT complex in which the FRT DNAs are still sequestered.
As indicated above, changes in the concentration of FLP protein will affect the partitioning of the intermediate produced by disassembly step 1. The breakdown of this intermediate to free products will be governed by a first order rate constant and depend on the concentration of the intermediate. The rate of the reverse reaction generating a fully populated complex by adding FLP protein to the intermediate will depend on the concentrations of both the intermediate and free FLP protein and be governed by a second order rate constant. Free FLP protein will produce a larger effect on the partitioning if the breakdown of the intermediate to free products is slow. The strong suppression of cross-product formation observed in this study suggests that the breakdown of intermediate to free products is at least partially rate limiting under normal reaction conditions.
We considered the possibility that FLP protein binds to a substrate and that this complex then binds to a second, unbound FRT substrate to initiate a reaction. Our experiments argue against this pathway for FLP-mediated reactions, since experiments in which unbound substrate is introduced into an ongoing reaction show the same lag in cross-product formation as experiments where two ongoing reactions (both with FLP protein present) are mixed (Fig. 7).
Based on binding studies done by Beatty and Sadowski(21) , one could envision a reaction path in which FLP protein binds to one FRT target, two such protein-DNA molecules come together to react, and the products of this reaction separate as two new FLP-FRT complexes, which go on to find new reactive partners. The experiments presented here argue against this reaction sequence and for a pathway in which most or all of the FLP monomers must dissociate before the FRT products are free to react with new partners.
The time course for the suppression of cross-product formation in the mixing reactions is similar to the rate of product formation in the control reactions (Fig. 6), indicating that the stable complexes are predominantly bound to products. This suggests that the chemical steps in a recombination reaction are fast relative to stable complex formation so that assembly and disassembly of complexes are both slow processes. Alternatively, FLP protein could bind rapidly to substrate DNA but form a stable complex only after recombination had occurred. In the latter case, the recombination pathway would involve conformation changes in the FLP monomers in the complex so that the products were bound differently (and more tightly) than the substrates. This would lead to a ``one-way enzyme'' (17) in which release of products would be rate limiting.
Jayaram and colleagues (6, 7) have shown that
domains from two different FLP monomers must come together to form a
single active site for DNA cleavage and strand exchange. Qian et al. (10) have demonstrated that protein-protein interactions across
the spacer region of the FRT are strong enough to form complexes with a
half-life of 1 to 2 h. Schwartz and Sadowski (8, 9) have shown that FLP protein promotes a sharp
bending of the FRT substrate in the course of catalyzing site-specific
recombination. These results, together with the data presented here,
reveal a protein-DNA complex that is held together very tightly and
cooperatively. The stepwise dissociation pathway outlined in this work
suggests further that at least one of the FLP monomers in a presumably
tetrameric complex is held less tightly than the rest. Recent work
indicating that three FLP monomers may be necessary and sufficient to
carry out a set of DNA cleavage and strand exchange reactions leads to the suggestion in Fig. 8that the
``loose'' component is a single monomer, and it seems
reasonable to hypothesize that three monomers would be sufficient to
continue the sequestration of the reaction products. Although we
emphasize that this proposal is not uniquely consistent with the data
in the current study, we presume that the only alternative is that two
FLP monomers remain after disassembly step 1, which would seem to be
the minimum required to sequester two FRT sites in the manner
demonstrated.