Synapsis of attachment sites during lambda integrative recombination involves capture of a naked DNA by a protein-DNA complex

Cell, Vol. 52, 9-17, January 15. 1966, Copyright 0 1966 by Cell Press

Synapsis of Attachment Sites during Lambda Integrative Recombination Involves Capture of a Naked DNA by a Protein-DNA Complex

Evelyne Richet,” Peter Abcarian,t and Howard A. Nash Laboratory of Molecular Biology National Institute of Mental Health Bethesda, Maryland 20892

Summary

During lambda integration, Int recombinase must specifically bind to and cut attachment sites on both the viral and host chromosomes. We show here by footprinting and by a novel cleavage assay that the bacterial attachment site, attB, cannot stably bind Int in competition with other DNAs. Instead, during recombination reactions, aftB obtains its Int by collision with the intasome, a nucleoprotein assembly that forms on the viral attachment site, affP. Our cleavage assay also shows that the capture of atfB by the attP intasome does not depend on DNA homology between the two sites; synapsis is governed solely by protein-protein and protein-DNA interactions.

Introduction

The integration of bacteriophage lambda into the chromosome of its E. coli host is accomplished by reciprocal recombination between sequences called attachment sites. The viral attachment site, attP, is known to span 240 bp and to encompass a collection of binding sites for the recombination proteins Int and IHF (reviewed in Weisberg and Landy, 1983). The binding of Int, a viral protein of M, = 40,000, to atfP is particularly complex (Figure 1). Int binds to the core of attP, the region where crossing-over takes place, recognizing a pair of inversely repeated binding sequences that are found at the junction of the core and the flanking arms of attP (Ross and Landy, 1983). Int also binds to sites that lie 50 to 150 bp away from the core, in this case interacting with a completely different sequence that is repeated five times in the arms of aUP (Ross and Landy, 1982). Chemical modification studies (Kikuchi and Nash, 1979; Ross and Landy, 1982) support the idea that Int is a bifunctional binding protein with a do- main devoted to interaction with arm sequences separate from that responsible for core binding. IHF, an E. coli protein of M r = 20,000, has three binding sites in the arms of attP (Craig and Nash, 1984). Thus, under recombination conditions, attP will be decorated with many copies of two different recombination proteins. Several lines of evidence make it clear that, under the influence of supercoiling, attP, Int, and IHF form an ordered structure, the intasome, that is the active species in integrative recombination (Echols, 1986; Richet et al., 1986).

* Present address. Unite de GBn&ique Mokculaire, lnstitut Pasteur, 28 rue du Dr Roux. 75724 Paris Cedex 15, France. TPresent address: Dartmouth Medical School, Hanover, New Hamp- shire 03756

By contrast with attP, atfB is short and simple (Figure 1). Within the 20 to 25 bp that comprise attB (Mizuuchi and Mizuuchi, 1985) no IHF binding sites are found, nor are there sequences of the type that direct Int binding to the arms of aftf? Furthermore, supercoiling of attB DNA is not important for the in vitro function of attB (Mizuuchi and Mizuuchi, 1979). attB contains the same 15 bp sequence that is found at the core of attP; this and a few bp of flanking DNA complete a pair of junction sequences for the binding of Int (Ross and Landy, 1983).

The binding of Int to the core of attachment sites is a critical step in recombination. When bound to junction sites, Int can act as a topoisomerase, cleaving the DNA pre- cisely at the positions of strand exchange and forming a covalent protein-DNA bond (Craig and Nash, 1983). This site-specific topoisomerase activity, which can be observed as an inefficient reaction on isolated DNA frag- ments, presumably mediates the strand exchanges that occur during a complete recombination reaction. In this view, the strand exchange step in recombination is a pro-

cess limited to Int protein bound at the core regions of artP and affB. It is known that within the intasome Int is bound tightly to the core of attP (Richet et al., 1986), but the source of Int for attB has remained questionable. Does attB bind Int from solution and thereby bring its own recombination protein into the reaction? Or does attB en- ter the reaction as a naked piece of DNA and receive its Int from the intasome? Indeed, we previously proposed (Richet et al., 1986) that the need for an ordered intrasome structure reflects the need for positioning Int promoters so as to capture attB. Determining the source of Int for attB is critical for understanding lambda integration because it defines the reactive species in the process. In this paper we present evidence that attB does not obtain its Int from solution; instead, synapsis involves the interaction between a naked piece of attB DNA and a protein-nucleic acid assembly formed at atff? Moreover, we show that capture of attB by the intasome does not involve homologous DNA-DNA pairing and thus depends solely on protein-DNA interactions. We suggest that the pattern of protein assembly on one DNA site followed by interaction of this assembly with a second DNA site may be important in other systems.

Results

Footprinting Recombination Reactions When an end-labeled fragment of DNA containing attB is mixed with Int, both nuclease and chemical protection studies have demonstrated a specific protein-DNA interaction (Ross et al., 1979; Ross and Landy, 1983). Thus it might seem argumentative to question whether attB can acquire its Int from solution. However, the protection studies indicate that the binding of Int to attB is very weak. For example, to achieve measurable protection of attB, Int concentration must be raised to a level at which nonspecific binding is a serious problem (ROSS and Landy,

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n an s

I

Figure 1. Binding Sites for Recombination Proteins on Attachment Sites

Int binding to the core and the arms of the attachment sites are indicated by stippling and ////, respectively. IHF binding is Indicated by \\\\. Coordinates of the attachment sites are given with respect to the center of the core, the 15 bp region that is common to all sites. In anP, regions to the left and right of the core are called P and P’arms, respectively. In the P’ arm there is a cluster of three tandemly repeated Int binding sequences (coordinates +55 to +85); the other Int and IHF arm binding sites each appear to be separate (Weisberg and Landy, 1983). On both attP and anB, a pair of inversely repeated Int core binding sites occupy coordinates -9 to -1 and +3 to +ll (Ross and Landy, 1983). Although not shown, Xis binds to a pair of sites that span coordinates -99 to -68 (Yin et al., 1985). Note that integration gener- ates recombinant attachment sites, atiL and attR, that contain subsets of the binding sites found in affP and attB.

1983). In all previous studies, the shorj attB fragment was the only DNA in the binding assay, but in order to under- stand recombination we must determine the state of attB when a second DNA is present. We therefore carried out

IHF: 0 47 47 47 47 Int: 0 376 188 94 47

0 47 47 47 47 0 376 188 94 47

1 2 3 4 5

lb1

chemical footprinting studies that employed nanomolar concentrations of both attB and a supercoiled attP partner and included Int and IHF proteins in amounts suitable for recombination. Since crossing-over between attP and attB would confuse our analysis by decreasing the amounts of these DNAs and by generating products that also bind Int, we prevented recombination by using an attP partner that had an altered DNA sequence in the 7 bp “overlap” region that lies between the Int cleavage sites. It is known that such altered sites, called saf mutants, bind Int normally but are unable to recombine with a wild-type attB although they readily recombine with an attB containing the identical saf alteration of the core (Weisberg et al., 1983).

Figure 2a shows the result of a typical chemical protection experiment. Over a wide range of Int concentrations, little or no interaction is observed with attB. In these experiments the DNA in the reaction mixture was unlabeled and attB was visualized by probing with a specific end- labeled oligonucleotide (Church and Gilbert, 1984). By using a different end-labeled oligonucleotide, one can visualize the attP saf partner present in the same reaction mixture. Figure 2b shows that over the entire range of Int concentrations tested, the core region of attP is occupied by Int. (As reported before [Richet et al., 19861, other Int and IHF binding sites in the arms of attP are also occupied.) In parallel, reactions were performed in which arts and an attP having the same overlap region were incubated with the identical concentrations of Int and IHF as used in Figure 2. Under these conditions, recombination

1 2 3 4

(cl

Figure 2. Footprinting Different Attachment Sites in Mock Recombination Reaction Mix- tures

Each reaction mixture contained a supercoiled circle carrying atiP safG and linear DNA frag- ments containing either a wild-type atti3 (a and b) or an attL with a wild-type overlap region but with a mutant IHF site (c). Protection of these DNAs (present at a concentration of 3-4 nM) by the indicated concentrations (nM) of Int and IHF against attack by dimethyl sulfate was carried out as described in Experimental Proce- dures. In (a), the reaction mixture was probed for the bottom strand of the atfB partner. Pro- tection of -9 (G) and enhancement of -10 (G) as well as more subtle changes are expected if Int binds to the core region (Ross and Landy, 1983). In (b), the same reaction mixture as shown in (a) is probed for the top strand of the attP partner. Strong protection of -7(G) and +8(G) are diagnostic of Int binding to the core. Int and IHF binding to other sites in a?fP can be inferred from alteration of other bases, as described in Richet et al. (1986). In (c), the reaction was probed for the bottom strand of the affL parent. Lanes 3 and 4 are purine and pyrimidine sequence markers, respectively. In lane 2, alteration by Int of core positions -9(G), -10(G), -6(G), -3(A), +9(A), and +13(G) is

evident. Binding of Int to the P’1, P’2, P3 sites in the P’ arm region of the attL can also be inferred from alteration at positlon +60(G), +68(G), +79(G), respectively.

Synapsis of Attachment Sites 11

was highly efficient, although the highest Int concentration caused significant inhibition of the reaction (data not shown). Thus, under actual recombination conditions, the core of attP is filled but the core of attB is naked. We have

confirmed the earlier reports (Ross et al., 1979; Ross and Landy, 1983) that Int can bind to an end-labeled attB fragment (data not shown). However, here too an attP safpart- ner successfully competes for binding. We have also noted that the binding of Int to atrB is so weak that even nonattachment site DNA is an effective competitor. For example, the binding of Int to an end-labeled attB is depressed to undetectable levels by the addition of salmon sperm DNA (100 flglml). Moreover, the vector por- tion of the unlabeled attB plasmid used in Figure 2 precludes a strong footprint even in the absence of an attP partner. In both cases (data not shown), either fortuitous Int binding sites or nonspecific interactions must be suffi- ciently strong to compete effectively with attB DNA. The finding that attB fails to show evidence of Int binding under recombination conditions suggests not only that it enters the reaction as a naked piece of DNA but that it cannot stably acquire Int through interaction with the intasome formed on the attP saf partner. In a later section, we present experiments showing that nonhomology between attB and attP saf does not prevent synapsis; capture must be evanescent.

A Tight-Binding Relative of affB There is an important caveat in applying footprinting results to proposals concerning the mechanism of recombination. Protection studies tell one only about the behavior of the bulk of DNA molecules in solution; minor species go undetected. Thus our data do not rule out the possibility that at any given instant a small fraction of attB DNA interacts with Int. One must therefore consider the hypothesis that, although the bulk of the attB population is naked, the intasome selects that small fraction that is complexed with Int. To address this question we tried to construct variants of atrB that bind Int tightly. We reasoned that if a naked arrB is the preferred substrate of integrative recombination, tight-binding variants should behave quite differently from the standard arrB.

We tried to improve the binding of Int to the core region by using attachment sites that have either extra IHF sites or extra Int sites. First, we used a segment of attP, containing the core and flanking IHF sites, as a replacement for arrB since earlier work has shown that truncated versions of arrP can serve as analogs of arrB, i.e., recombine with a supercoiled arrP partner (Mizuuchi and Mizuuchi, 1980; Hsu et al., 1980). We hoped that either the tighter binding of Int to the arrP core (Ross and Landy, 1983) or, more likely, the presence of flanking IHF binding sites (Craig and Nash, 1984; Bauer et al., 1986) would improve the binding of Int to the core of this analog. But footprinting studies demonstrated that, just as with arts, the core region of the variant (plasmid pMJB11 [Ross and Landy, 19831, containing attP sequence from positions -70 to +46) appears naked in the presence of an attP saf partner

(data not shown). Moreover, at high concentrations of IHF, the analog recombines with supercoiled attP much less

efficiently than does a standard attB. We believe that IHF binding to this DNA is not only unable to dramatically stabilize the binding of Int but actually interferes with its capacity to be captured by the intasome, perhaps because of simple steric interference.

We next turned to an analog of affB that has Int arm binding sites in addition to the core binding sites. This is conveniently provided by attL (Figure 1). To avoid interference by IHF binding, we used a variant of arrL in which the lone IHF binding site had been inactivated by the QH’ mutation (Gardner and Nash, 1986). As shown in Figure 2c, this variant is able to bind Int to its core (in addition to its arm) even in the presence of an arrP saf partner. In other experiments (not shown), we have separately probed such recombination mixtures for both the affP and the attL variant; both showed strong occupancy of their core regions by Int. Since the attL derivative we have used does not bind IHF and since its capacity to bind Int to its core does not depend on the presence of an affP partner (data not shown), we conclude that Int bound to the arm of attL must help core binding. This could reflect protein-protein interactions between separate Int protomers that are bound to the two segments. Alternately, since Int is a bifunctional protein (see above), one Int protomer might be shared between both arm and core sites. Despite our ignorance of the stoichiometry of binding, the capacity of attL to bind Int reasonably tightly to its core allows us to ask whether this attachment site behaves as a simple analog of arrB or whether covering the core with Int con- verts this DNA into a novel type of attachment site.

Figure 3 presents several cases in which the tight- binding variant recombines differently from arrB. In each case, we find that conditions that are unsuitable for recombination of a standard attB are permissive for the tight- binding variant. Thus, in agreement with earlier work, affB fails to recombine when IHF is omitted (Figure 3a), when supercoiled attP is replaced with linear attP (3b), and when supercoiled atiP is replaced with either a supercoiled (3c, 3d) or a linear (3e) wild-type arrL. Each of these alterations interferes with intasome formation (Richet et al., 1986); the concurrent failure of recombination confirms the assertion that the intasome is essential for recombination between affP and atis. By contrast, when arrB is replaced by the arrL QH’variant, significant recombination occurs under conditions where an intasome cannot form (Figures 3a-3e). The same disparity in recombination potential of attB vs. the arrL variant is seen (data not shown) when the recombination partner is a supercoiled attP derivative that lacks either the Int binding site at -140 (Bauer et al., 1986) or the IHF binding site at -120 (Gard- ner and Nash, 1986). To quantitate and increase the sensi- tivity of our assays, we repeated the experiment of Figure 3 using an end-labeled artB or affL variant as substrate. Recombination of the labeled artL variant under the condi- ions indicated in Figures 3b, 3c, and 3e was at least 30

times as efficient as was recombination of attB. Recombi- nation of the labeled attL variant in the absence of IHF (as in Figures 3a and 3d) was reproducibly less robust but still was in marked contrast to the undetectable recombination of arrB under these conditions. Thus both quantitatively

Cell 12

SCP x

tin P x

SC L SCL lin L x x x

B L B L B L B L B L

IHF - - Int , + + ,

(al (bl

+ + + +

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- - + + + + L+ +,

IdI (al

Figure 3. Comparison of attB and an attL Variant as Recombination Partners

Each panel shows an ethidium bromide-stamed agarose gel of a recombinatton reaction; recombinant products, where they can be detected, are marked by an arrowhead. The two partners in each reaction are Indicated above the figure. The partner in the top line was a plasmid containing either a wild-type atfP (lanes a, b. f) or a wild-type attL (lanes c, d, e). As Indicated, these plasmlds were provided either as supercolled circles or as linear molecules cleaved at a unique EcoRl site. The second partner in each reaction was either a singly cut plasmid containing attB or a singly cut plasmid containing an attL that had a damaged IHF site. Int was included at 125 nM, and IHF was etther omitted or included at 15 nM. The remainder of the recombination reactlon conditions are described in Experimental Procedures.

and qualitatively, the tight-binding variant displays novel

properties and is not a close analog of attB. It appears that naked and covered cores have different recombination potentials, and we believe they recombine by different pathways.

Unlike the inequality between attP and attB partners in the standard integrative recombination reaction, covered cores seem to recombine as equal partners since two attLs can recombine with one another even in the absence of IHF or supercoiling (Figures 3d, 3e). This mode of recombination may be operative in vivo, since E. coli that are mutant for one of the subunits of IHF retain a substantial capacity to carry out recombination between two copies of atiL (Kikuchi et al., 1985). The behavior of this “covered pathway” is reminiscent of simpler recombination systems such as the Cre-promoted recombination of Pl lox sites (Hoess and Abremski, 1984). It would be in- teresting to determine whether the topological conse- quences of attL x attL recombination reflect the simplicity of this pathway. In any case, such a pathway must be dis- tinguished from that used during lambda excisive recombination since, under our conditions, recombination between the attL variant and artfl still requires Xis protein (Gardner and Nash, 1986). It may be that there are many pathways to synapsis; that used by arts places special de- mands on its partner because the site has such a poor affinity for Int. We must point out, however, that we do not know if the different recombination potential of attL is due solely to the occupancy of its core. Because we have been unable to find conditions in which the arm of attL is occupied by Int while the core remains empty, we cannot rule out the possibility that the novel properties of this site simply reflect the presence of Int bound to the arms. Nevertheless, because the core is the critical region for

strand exchange, we favor the interpretation that the binding of Int to the core is the key factor in determining the behavior of attL.

We have considered the possibility that the expanded repertory of recombination reactions observed when the attL variant replaces attB simply means that attL is just a more effective version of attB. If this were so, it would argue that the reactive form of attB is not a naked piece of DNA but one that is covered with Int. However, Figure 3f shows that the capacity of the attL variant to recombine with the standard integrative recombination substrate, supercoiled atfP, is not substantially greater than that of attB. This is a sensitive test of the relative efficiencies of attB vs. attL since under our standard recombination conditions, attachment site concentration is limiting and the two sites behave equivalently throughout the time course of recombination reactions (data not shown). We suspect that, rather than being a more effective substrate, the capacity of attL covered with Int to recombine with the intasome is low. Some of the recombination between supercoiled attP and attL seen in Figure 3f may be due to recombination of a covered attL with artP molecules that have bound Int but are not wrapped into an intasome. Moreover, some of this recombination may be due to a fraction of the attL substrate that is not completely covered by Int. In any case, attL is no better at recombination with supercoiled attP than is attB, a result that argues against the possibility that attB covered with Int is the preferred substrate for the intasome.

:

A Cleavage Assay for Int Binding to attB Footprinting methods all suffer from the uncertainty that the reagent used to probe for binding of a protein to a DNA site may perturb that binding. This concern is especially


att L +/e

Int: - +

(a)

appropriate for weak binding sites like those found at arrB. Thus one must question to what extent the apparent nakedness of arrB seen in Figure 2 reflects its state in mixtures that are not exposed to dimethyl sulfate. We have found that some recombination can take place in the presence of dimethyl sulfate (data not shown), so the putative alteration caused by the reagent cannot be drastic. Nevertheless, one would like to assess binding of Int to attB without an external probe. Moreover, since the interaction of Int with attB appears to be weak, one would like to assay this binding in a way that reports even a transient association. These two goals have been achieved by using the topoisomerase activity of Int.

As stated in the introduction, Int bound to junction sites can cleave DNA. As usually assayed, only a very low level of cleaved products accumulate (Craig and Nash, 1983). This is because Int topoisomerase reseals the broken DNA very efficiently so that, at the moment the reaction is deproteinized, only a tiny fraction of the DNA is caught between the acts of breakage and reunion. As diagram- med in Figure 4a, this situation changes with DNA substrates in which the region between the Int cleavage sites is not perfectly Watson-Crick paired. With such a heteroduplex substrate, rejoining of the broken ends fails to occur and strand breaks accumulate. Nash et al. (1987) have shown that Int binding to attL heteroduplexes results in a high proportion of double-strand breaks (single-strand breakage was not studied in detail but appears to be a mi- nor product). We have now applied the same assay to attB heteroduplexes. As seen in Figure 4b, an affB heteroduplex (saf+/safG) is cleaved much less efficiently than is the corresponding attL heteroduplex. The cleavage effi-

- + Heteroduplexes were made by annealing com- plementary strands from a wild-type attachment site, and a variant site s&G, whose overlap sequence differs from wild-type in 3 of the 7 base pairs. (a) When Int binds to the core region of such a heteroduplex, Its topoisomerase domaln cuts the overlap sequence and cova- lently joins to one broken end. Resealing of this break is hindered by the noncomplementarity of the overlap sequence. Although the diagram pictures double-strand cleavage as occurring in two discrete steps, the degree of coupling between the cuts is not known. As revealed by

. comparlng the sizes of native and denatured molecules, after double-strand breaks are made, Int efficiently catalyzes the joining of sister

3 4

4 strands to form hairpins (H. Nash, unpublished data). (b) 5’ end-labeled affL or attB saf+/safG heteroduplexes were mixed with 12.5 pglml of salmon sperm DNA and incubated in the presence of 94 nM IHF and 75 nM Int as described in Experimental Procedures. The mixtures were electrophoresed through a native acryl- amide gel (5%) and autoradiographed. In lanes 2 and 4, the position of the cleaved products are marked with arrowheads.

att B Figure 4. Cleavage of Heteroduplex Attach-

+/e ment Sttes

ciency thus confirms the conclusion based on footprinting studies that arrB is unable to compete for Int even with the nonspecific carrier DNA that is included in these reactions. We have tried a second attB heteroduplex substrate (safG/saf+) and a wide variety of Int and IHF concentrations; under no conditions do we find substantial cleavage of arts.

It occurred to us that attB heteroduplexes might provide a useful way to assay the capture step in recombination. During this step, the intasome is believed to provide the junction sites of arts with Int. Thus capture of a heteroduplex attB should result in its cleavage, an easily assayed event. Heteroduplex arts constructs have been used successfully as efficient substrates for in vitro site- specific recombination (Bauer et al., 1984, 1985; Nash et al., 1987), an observation meaning that the heteroduplex bubble is not an impediment to any step in recombination, in particular, attB capture. We reasoned that even if one introduced a second alteration that blocked a late step in recombination such as strand exchange, one might still detect the earlier step of capture by looking for cleavage of arrB. Accordingly, we mixed attB heteroduplexes with IHF, Int, and variants of arri? The attP variants had alterations in the sequence of the overlap region that rendered them nonidentical to either of the strands in the atiB heteroduplex. As mentioned earlier, with homoduplex substrates, the overlap sequence of attP and arrB must match perfectly to generate completed recombinants (Weisberg et al., 1983; Bauer et al., 1985; deMassy et al., 1984). Fig- ure 5, lanes 1 to 4, show that when an attB saf+/safG heteroduplex is incubated with IHF, Int, and any of a series of supercoiled circles containing attP saf variants, there is

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attP saf attP saf + -I K u,

‘-2 0 +1 0 i% g Mn I

123456789 Figure 5. Cleavage of aft6 Heteroduplexes Is Stimulated by affP

End-labeled aft6 saf+/safG heteroduplexes were mixed with plasmid DNA and incubated with 94 nM IHF and 125 nM Int as described in Expenmental Procedures. The plasmid DNAs in lanes 1 to 4 were supercoiled crrcles contarmng, respectively, affP saf-2G, atfP safOA, atfP saf+lG, anP saK)C. Each saf variant has a single base pair alteration from the wild-type sequence (described in detail in Experimental Procedures). The plasmid DNAs in lanes 5 to 7 vyere supercoiled circles containing wild-type atrL, wild-type attR, or no attachment site, respectively. The plasmid DNA in lanes 8 and 9 contained, respectively, singly cut or supercooled circles bearing aftP saf+. In thus case, since the overlap region of affP perfectly matches one of the strands of the heteroduplex, recombination with the supercoiled partner is efficient (Bauer et al., 1984, 1985). One of the recombinant products (attL) is heteroduplex and is efficrently cut by Int (Nash et al., 1987). The posr- tion of affB (BOB’), atiR (POE’), attL (BOP), and the cleaved products (6 and 6’) are marked.

a dramatic stimulation of the cleavage of the affB heteroduplex over that seen in the absence of a partner (Fig- ure 4b). The same result is observed in a series of experiments that use attB safGlsaf+ heteroduplexes (data not shown). The amount of cleavage in these experiments is at least as great as the amount of recombinant produced in a standard in vitro cross. We conclude that capture of MB by attP does not require homology between the sites. Note that each nonhomology that we have tested is sufficient to block the formation of completed recombinants (Figure 5). Thus the requirement for homology must be restricted to a step after synapsis.

To challenge the hypothesis that the cleavage of a heteroduplex attB reflects its capture by the atfP intasome, we replaced attP by a series of variants that are unable to form an intasome (Richet et al., 1986). Figure 5, lanes 5 to 8, shows that when supercoiling or either the left or right

arm of affP is removed, no cleavage is seen above the background observed when a heteroduplex affB is incubated in the presence of nonspecific DNA. In other experiments (not shown), we replaced the affP partner by an attP that contained a mutation inactivating either the IHF binding site at -120 (Hl), the Int binding site at -140 (Pl), or the Int binding site at +80 (P’3). Each of these alterations depresses recombination in standard crosses (Bauer et al., 1986; Gardner and Nash, 1986) and each should

disrupt the intasome. As expected, in all these cases, cleavage of an attB heteroduplex was greatly reduced.

Discussion

We have used three kinds of experiments to explore whether the bacterial site for lambda integration enters the recombination reaction with Int recombinase already bound to it or whether attB must acquire its recombinase from that associated with the viral site. First, chemical protection studies showed that Int cannot stably bind to aRB in the presence of a second DNA; the Int binding sites on attB are apparently so weak that even nonspecific DNA successfully competes for Int. A second series of experiments examined double-strand breaks made by Int on attachment sites that contained noncomplementary pairs within the core region; in these experiments, Int acts as its own probe for the efficiency of binding. Cleavage was scarcely detectable on attB heteroduplexes, again indicat- ing very weak binding of Int. In contrast, heteroduplexes of attL, a site that shows strong Int binding in footprinting studies, are efficiently cleaved by Int. A third line of evidence compared the recombination behavior of the weak- binding attB with that of the tight-binding attL. The two sites appeared to recombine by different pathways; attB required a partner that could form a complete intasome but the artL variant could recombine even in the absence of supercoiling, in the absence of IHF, or with partners lacking binding sites that are essential for intasome formation. Each kind of experiment therefore suggests that the reactive form of attB, i.e., the form that encounters attP, is naked DNA. We recognize that our experiments are in- direct, and that each result could be interpreted differently. Thus the cleavage studies depend on the assump- tion that binding of Int to the core is sufficient for cleavage. It could be argued that Int binds stably to attB but fails to cleave unless an allosteric transition is made that depends on contact with a# While this possibility is refuted by our footprinting studies, both footprinting and cleavage studies leave open the possibility that the reactive form of attB is the small fraction to which Int is bound. Our recombination studies argue against this possibility, but the differences in recombination potential between the attL variant and attB could be solely attributed to the presence of arm binding sites in affL rather than to its capacity to bind Int tightly to its core. Although we acknowledge these uncertainties, we are impressed that the simple postulate that affB is naked provides a cogent explanation for all our data, a collection of experiments that rely on a variety of protocols and that examine different aspects of protein-DNA interactions. A more direct demonstration that atfB obtains its Int from the intasome would require isolation of this structure free of unbound proteins. While we have not yet isolated the intasome, we have observed that integrative recombination persists in the presence of a large excesSof nonspecific DNA (E. Richet, unpublished data). Under these conditions affB continues to recombine with normal efficiency, although we expect all the Int not specifically involved in the intasome to be segregated to the carrier DNA. We believe that, taken together, our experiments establish the concept of a naked attB interact-


Figure 6. A Proposed Pathway to Synapses during Integrative Recom- bination

atrP is represented by a ribbon; this DNA is pictured (top panel) to be complexed with Int and IHF. To represent the proposal that this rmtial structure is not highly condensed (Echols, 1986; Rrchet et al., 1986) the complex is shown to be organized into two blobs, eachcontaining both Int and IHF, but the two parts of the complex are not in close contact. Supercoiling helps to organize the entire complex into a more compact structure, the rntasome (middle panel). The intasome is the species that interacts with aft6 (hatched ribbon; bottom panel). In this synaptic step, some of the Int protomers that make up the intasome bind to the junctron sites of affS. Int topoisomerase cleavage then inrti- ates the exchange of DNA strands (not shown).

ing with the attP intasome as the favored hypothesis for the pathway of lambda integrative recombination.

The insight gained from this work into the state of MB coupled with previous results on the organization of attP (Richet et al., 1986) permit us to sketch an outline of the early steps in recombination (Figure 6). The process be- gins by the binding of Int and IHF to their multiple sites on attP Although the initial binding may produce an open structure (Figure 6, top), to be active in recombination this assembly must fold into a compact and ordered structure, the intasome (Figure 6, middle); such folding is dependent on supercoiling. Synapsis is achieved (Figure 6, bottom) when a naked piece of DNA bearing attB collides with the intasome, and Int protomers bound to attP interact with the junction sites present on the bacterial partner. Since Int can recognize either of two different DNA sequences (Ross and Landy, 1982, 1983) it is tempting to speculate that one (or more) of the Int protomers bound to an arm site uses its unoccupied core binding site to catch attB. However, it has not been demonstrated that one Int protomer can bind two pieces of DNA simultaneously. Other possibilities for capture could arise if the intasome included Int protomers that were not directly attached to attP sequences, but were held in the structure by purely protein-protein interactions.

It is important to point out that up to this step we have not invoked DNA-DNA pairing. It is known (Weisberg et al., 1983) that some step in integrative recombination requires perfect homology between the overlap region of atiP and attB (but not in the segment of the core that lies outside the region; Bauer et al., 1985). However, the data

in this paper argue strongly against homologous pairing of DNA as a requirement for any step in recombination up to and including synapsis. We assessed synapsis by the capacity of attP to stimulate the cleavage of affB heteroduplexes; in this assay we find that there is no need for identity between the DNA of the overlap region of atrP and that of attB. This result virtually eliminates what had once been our favorite hypothesis for the role of homology in recombination, i.e., the juxtaposition of attachment sites by the homology-dependent construction of a segment of four-stranded DNA (Kikuchi and Nash, 1979; Nash and Pollock, 1983). It has recently been shown that in certain crosses involving attachment sites with nonidentical overlap regions, although no completed recombinants are produced, Holliday structures accumulate (Kitts and Nash, 1987; Nunes-Dtiby et al., 1987). These experiments also indicate that synapsis does not require two perfectly matched overlap regions, but they leave open the possibility that DNA pairing at the left of the overlap region could be needed for synapsis. This is because crosses between partners that are not homologous in this segment accumulate neither Holliday intermediates nor completed recombinants. The results presented here (Figure 5) show that the capture of attB occurs regardless of the location of the nonhomology. Equally efficient capture is seen when nonhomology is present in positions compatible (+l) and incompatible (0, -1, -2) with Holliday structure formation. We conclude that homology is needed only after synapsis, i.e., during the strand exchange step in recombination. Kitts and Nash (1987) discuss various ways in which homology could be used in strand exchange and indicate a preference for a model in which resolution of the Holliday junction requires migration across the overlap region, a process that is thought to depend on homology. In any case, our current results imply that synapsis is achieved by a combination of protein-protein interactions and protein-DNA interactions. If direct contacts between DNA partners occur during synapsis, they do not involve sequence-specific interactions.

After infection of E. coli by bacteriophage lambda, lysogeny requires that the viral and host sites find each other. According to the evidence we have presented, this problem is like that seen in many simpler protein-DNA interactions, such as the formation of operator-repressor complexes, in which a naked DNA sequence must be identified by a site-specific binding element. Of course, the attP intasome is a more complicated binding appara- tus than has been studied before. Nevertheless, we think it likely that mechanisms such as sliding and hopping along the target DNA that are used by elements like lac repressor to locate their unique binding sites in the E. coli chromosome (Berg et al., 1982) will be used by the intasome in the process of integration. Kinetic tests of these possibilities should be fruitful.

The strategy of capture of a naked DNA by a protein-nu-

Cell 16

cleic acid assembly may not be rare. There is convincing evidence that two lac operators can be bridged by lac repressor (Kramer et al., 1987; Borowiec et al., 1987). Since the native form of lac repressor appears to be a tetramer with two binding sites for DNA, it is possible that bridging is accomplished by the binding of the tetramer to one site followed by capture of the second. A second example of the kind of pathway we have proposed for site- specific recombination may be found in general recombination systems. Here, synapsis of homologous partners appears to be achieved by the interaction of a naked double-stranded DNA with a nucleoprotein filament (Kahn and Radding, 1984; Harris and Griffith, 1987). Derbyshire et al. (1987) have recently provided evidence that the transposition of the prokaryotic element IS903 is initiated by the assembly of the transposase onto one end of the element: the other end of the element, free of protein, then interacts with this assembly. (A similar scheme has also been suggested for the transposable element TnlO [Way and Kleckner, 19851.) The two ends of IS903 are com- posed of identical sequences but Derbyshire et al. (1987) propose that they are made inequivalent by their different proximity to the transposase gene, which loads preferen- tially onto the nearer site. There may be other examples of apparently identical recombination sites functioning in an asymmetric way. We are impressed that in several con- servative site-specific recombination systems that are thought to comprise two equal partners, alteration of one partner site reduces recombination much less than when both partners carry the same mutation (Kitts et al., 1983; Abremski and Hoess, 1985). In some of these cases, the capacity of a wild-type site to rescue a mutant one may reflect an intrinsic asymmetry with which the sites are normally used. A particularly well-documented case of the asymmetric use of identical sites is found in the packaging of phage lambda virions (Feiss and Becker, 1983). Finally, we speculate that elements like eukaryotic enhancers and the promoters they regulate may sometimes use the strategy we have described for lambda integration. That is to say, protein factors may assemble on one binding site, say the enhancer element, and only then interact with promoter elements that are free of protein. Our work has shown that the mere demonstration that each element can bind a specific protein should not be taken as evidence that the pathway for interaction is by each DNA site first binding its protein and then the two protein-nucleic acid complexes coming together. Instead, we have learned that simple footprinting may be misleading in this regard and that a more realistic pathway may involve binding of all the proteins to one site followed by capture of the second.

Experimental Procedures

DNA and Proteins The origin of derivatives of pBR322 containing atfP (-251 to +242), attB (-970 to +770), atrL (-970 to +242), or attR (-251 to +770) IS given in Richet et al. (1986). Derivatives of pBR322 containing attL (-250 to +242) with either a wild-type IHF site or one InactIvated by a 4 bp change in IHF consensus sequence (the ClH’mutatlon) are described in Gardner and Nash (1986). Supercoiled plasmid DNA was prepared as described III Nash and Robertson (1981). lsolatlon and annealing of single strands of Ml3 derivatives containing atfL (-970 to +242) or attB (-570 to +390) to produce heteroduplexes followed es-

tablished procedures (Baueret al., 1985). Subsequent purification and labeling were carried out as described by Nash et al. (1987) except that the attB heteroduplexes were removed from vector sequences by digestion with EcoRl and Hindlll restriction nucleases. The overlap region of wild-type attachment sites extends from coordinates -2 to +4 (the origm of the coordinate system is the center of the core); the sequence of the top strand of this region reads 5’.TTTATAC-31 The safG mutation (Weisberg et al., 1983) changes this sequence to 5’- TTTTCAA-3! Other saf mutations used in this work are named by the position of the alteration and the mutant base at that position. saftlG (5’.TTTGTAC-31 was provided by J. Gardner, and the remalnmg mutants have been described (Bauer et al., 1985; Kitts and Nash, 1987).

Int and IHF were purified to near homogeneity as described previously (Nash and Robertson, 1981; Nash, 1983). Immediately prior to use, the proteins were diluted into a solution containing 50 mM Tris-HCI (pH 7.4), 10% glycerol, 2 mglml bovine serum albumin, and KCI at various concentrations.

Footprinting and Recombination Assays A standard reaction (0.025 ml) was inltlated by mixing, in order, 12.5 pl of 100 mM sodium cacodylate (pH 8.0), 0.83 VI of enzyme diluent containing 800 mM KCI, 6.0 ~1 of a solution of 10 mM Tris-HCI (pH 8.0) plus 1 mM sodium EDTA containing 0.25 pg each of two plasmid DNAs, and 3.12 PI of 40 mM spermidine. The components of this mixture and their order of addition were chosen so as to reduce the likelihood of precipitation of DNA by spermldlne. To this mixture was added 1.25 pl of a dilution of IHF adjusted to a final KCI concentration of 266 mM. The reaction was started by addition of 1.25 ~1 of a sample of Int that had been mixed with enzyme diluent containing 600 mM KCI. The reactions were incubated at 25%.

For footprinting assays, after 25 min the mixtures received 0.045 volumes of a freshly made Is-fold dilution of dimethyl sulfate (DMS; Al- drich Chemical Co.) in 95% ethanol. One minute later, 0.25 volume of DMS stop solution (Maxam and Gilbert, 1980) was added. Each sample was then analyzed as described in Richet et al. (1986). Briefly, the samples were deproteinized, restricted, and heated to introduce strand breaks at the position of methylated bases. They were then electrophoresed on a denaturing gel, transferred to a nylon membrane, and hy- bridized with an end-labeled oligonucleotide according to the proce- dure of Church and Gilbert (1984). Typically, all the DNA from a standard reactlon was loaded onto a single lane; footprinting assays were usually carried out at three to five times the standard scale to provide enough material for repeated gel electrophoresis. In a typlcal footprinting assay, the atfP partner carried a safG overlap sequence and the other partner had a wild-type overlap sequence. The abP partner was probed with ollgonucleotides as described in Richet et al. (1986). The other partner was restricted with Hhal before introduction into the reaction mixture and probed with oligonucleotldes that were com- plementary to either posltion -83 to -64 (top strand) or -81 to -62 (bottom strand).

For recombination assays, both partners contained the wild-type overlap sequence. The attB partner (or its attL QH’ analog) was digested with either Hhal or Sall before introduction Into the reaction mtxture, with equivalent results. After 40 min at 25’%, the reactions were quenched and electrophoresed In agarose as described by Nash (1983).

Cleavage Assays The standard reaction (0.02 ml) contalned 45 mM Trls-HCI (pH 8.0). 0.5 mM sodium EDTA. 5 mM spermldme. 70 mM KCI, 12.5 Kg/ml of plasmld or carrier DNA, 1000 to 3000 cpm of end-labeled heteroduplex DNA, IHF, and Int. The reactlons were Incubated for 1 hr at 25OC, adlusted to 10 mM magnesium chlortde, and digested for 1 hr at 37% with EcoRl and Aval restrIctIon nucleases. Restriction of these samples is de- signed to reveal recombinant products; cleavage of attB heteroduplexes by Int requires neither incubation at 37OC nor the presence of magnesium. After restrtctlon, the samples were electrophoresed at 2 V per cm through a native polyacrylamlde gel (5%) and autoradiographed _

Acknowledgments

We thank Carol Robertson for excellent technlcal assistance. Gerald Zon for oligonucleotlde synthesis. and Mamta Vasudeva for preparing

Synapses of Attachment Sites 17

the manuscript. We are grateful to Michael Brurst, Paul Kitts, and Olrvrer Raibaud for their comments on this manuscript. E. Rtchet was supported by the Centre National de la Recherche Scientifique and by the European Molecular Biology Organization. P. Abcarian was supported by the Medical Research Scholar Program of the Howard Hughes Medical Institute.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received October 13, 1987.

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Synapsis of attachment sites during lambda integrative recombination involves capture of a naked DNA by a protein-DNA complex