Site-specific recombination of bacteriophage lambda

J. Mol. Biol. (1983) 170, 19-38

Site-specific Recombination of Bacteriophage Lambda The Change in Topological Linking Number Associated with Exchange of D N A Strands HOWARD A. NASH AND THOMASJ. POLLOCKt Laboratory of Neurochemistry National Institute of Mental Health, Bethesda, Md 20205, U.S.A. (Received 9 March 1983) The changes in supercoiling that accompany site-specific recombination have been measured. In each experiment, the substrate was a circle that contained two attachment sites oriented as an inverted repeat; recombination between the sites inverts one segment of the circle with respect to the other. Using conditions developed in the accompanying work, a measurable amount of the recombinant is in the form of unknotted, simple circles. The difference between the topological linking number of this product relative to that of the substrate can be determined directly from the change in mobility during agarose get electrophoresis. With partially supercoiled substrates, both integrative and excisive recombination are characterized by a unique change in linking number, a relaxation of two topological turns. For excisive recombination, it has been possible to study closed circular substrates that lack supercoils. In this case, changes in linking number of both +2 and - 2 are observed. These results are used to evaluate various proposals for synapsis and strand exchange in bacteriophage Iambda site-specific recombination.

1. Introduction The site-specific recombinations t h a t integrate bacteriophage lambda DNA into the Escherichia coli chromosome and excise the inserted prophage are conservative reactions (Campbell, 1981; Nash, 1981). Studies of cells infected with lambda showed t h a t replication is not required for efficient site-specific recombination (reviewed by Weisberg et al., 1977). On this basis, lambda I n V p r o m o t e d recombination was judged to occur by a breakage and reunion mechanism. Recombination in cell-free systems has confirmed this conclusion: integrative and excisive recombination take place readily in the absence of deoxyribonucleotides and replication proteins (Mizuuchi et al., 1978; Weisberg & Landy, 1983). Thus, degradation and resynthesis of DNA are not an intrinsic part of the recombination mechanism. Studies in vitro have demonstrated a second way in which lambda site-specific recombination is a conservative reaction; the bond energy of the phosphodiester backbone is conserved during the breakage and reunion t h a t accompanies strand exchange. This assertion follows from the t Present address: Syntro Corporation, 11095 Torreyanna Road, San Diego, Calif. 92122, U.S.A. 19 0022-2836/83/290019-20 $03.00/0 © 1983 Academic Press Inc. (London) Ltd.

20

H.A. NASH AND T. J. POLLOCK

observation that continuous double-helical recombinants are produced in vitro from reaction mixtures that lack high energy cofactors like ATP (Mizuuchi et al., 1978). If the high energy phosphodiester backbone had been hydrolyzed during the breakage step of recombination, an energy-requiring ligation step would be needed to reseal the DNA backbone. The absence of such cofactors implies that the breakage and reunion is carried out by a topoisomerase, i.e, by an enzyme that forms a high energy covalent intermediate with the broken DNA and thus can reversibly break and reseal DNA strands. Int protein has a topoisomerase activity that can be detected uncoupled from its recombination activity (Kikuchi & Nash, 1979a). It has been shown that this activity can cleave attachment site DNA precisely at the position of the recombination crossover (N. Craig & H. Nash, unpublished observations). In this paper, we address a third property conserved during recombination: supercoiling. Earlier studies examined the relationship between the superhelical density of recombinant product relative to that of substrate circles (Mizuuchi et al., 1978,1980a). It was found that the product had a superhelical density that could not be distinguished from that of the substrate. However, the methods used in these early experiments, cesium chloride/ethidium bromide density gradient analysis and agarose gel electrophoresis, could not have detected a loss of 15 to 2 0 o of the initial supercoiling. Since these experiments were carried out with a large circular DNA (approx. 45,000 base-pairs), a loss of as many as 50 topological turns would have gone undetected. In this work, we have devised a novel strategy for comparing the superhelicity of recombinants relative to that of substrates. This method has permitted us to determine the precise change in topological linking number caused by recombination. We find that both integrative recombination and excisive recombination are accompanied by a characteristic change in topological linking number. The size, direction and uniqueness of this change are discussed in terms of alternate models for the mechanism of synapsis and strand exchange steps in recombination.

2. Experimental Strategy Lambda site-specific recombination takes place within a 15 base-pair sequence (the core) that is common to the phage, bacterial and prophage attachment sites (Landy & Ross, 1977). When two attachment sites are situated on a circle of DNA so that their cores form an inverted repeat, intramolecular recombination between the two sites inverts one segment of the circle with respect to the remainder (Mizuuchi et al., 1980b). For this kind of recombination, the product and substrate are circles of identical size and nucleotide composition. Therefore, barring complications from knotting (see below), any differences in etectrophoretic mobility between substrate and product must be due solely to a change in supercoiling. To simplify detection of this change, we use as substrate a single topoisomer species purified by electrophoresis from a population of substrate circles. The change in topological linking number of the recombinant product relative to this substrate is quantified by comparing the electrophoretic separation between substrate and product with the separation observed between

)~ SITE-SPECIFIC RECOMBINATION

21

individual topoisomers contained in the original population of substrate circles. The key element in this method is t h a t only relative changes in topological linking n u m b e r need be measured and these can be read directly from a gel by counting the n u m b e r of topoisomer bands separating substrate and product. In contrast, if recombination were to be assayed by an intermolecular reaction t h a t unites two circles into a composite, the electrophoretic mobility of the p r o d u c t would reflect a change in size as well as a change in supercoiling. To separate these two variables, the linking n u m b e r of all three circles, product and both substrates, would have to be determined and the change in linking n u m b e r calculated by subtraction. The measurement of the linking n u m b e r of a circular species involves several experimental steps and a curve-fitting analysis (Depew & Wang, 1975; Shure & Vinograd, 1976). I t seems likely t h a t experimental errors involved in the determination of three such values would lead to significant uncertainty in the final calculated answer. The same difficulties apply to intramolecular recombination between a t t a c h m e n t sites oriented on a circle as a direct repeat. Only inversion yields recombinants identical in size to substrates and thereby permits the direct determination of the change in linking number. There are, however, difficulties with the inversion strategy. As described in detail in the accompanying paper (Pollock & Nash, 1983b), the recombinant products of inversion are often k n o t t e d circles. Since knotting alters the electrophoretic behavior of DNA in an unpredictable way, no meaningful comparison can be made between the mobility of a knotted recombinant relative to t h a t of its u n k n o t t e d substrate. K n o t t i n g follows from the interwrapping of parts of the substrate circle; this interwrapping can be limited by reducing superhetical density and reducing the separation between a t t a c h m e n t sites. In this study, we have used a combination of these two factors to ensure t h a t a significant fraction of the recombinant products are simple circles. This has enabled us to make a direct comparison between the linking n u m b e r of parental and recombinant DNA. 3. Materials and Methods (a) Plasmids and proteins Plasmid pBP90A6 is an 8.4 kb¢ circle containing attP and attB oriented so that their homologous cores form an inverted repeat. The derivation of this plasmid from pBP90 (Mizuuchi et al., 1980b) is described in the accompanying paper (Pollock & Nash, 1983b). Plasmid pLR90A6 is the product of integrative recombination of pBP90A6. Both plasmids were grown in strain 204, labeled with [3H]thymidine and purified after chloramphenicol amplification as described (Nash & Robertson, 1981). Int and IHF were purified as described (Kikuchi & Nash, 1979b; Nash & Robertson, 1981). Purified Xis was a gift from S. Wickner and J. Auerbach. Purified HeLa cell topoisomerase I was a gift from Leroy Liu. (b) Relaxation of plasmid DNA Partially relaxed circles were prepared by treatment of supercoiled DNA with purified topoisomerase in the presence of varying amounts of ethidium bromide. A typical reaction (2-0 ml) was carried out in a polypropylene tube and contained 50 mM-Tris- HC1 (pH 7-4), Abbreviations used: kb, 10a bases; bp, base-pair.

-2"2

H. A. NASH A N D T . J. POLLOCK

120 mM-KCI, l0 mM-MgCl 2, 1.2 raM-potassium phosphate, 0.5 mM-sodium EDTA, 0.5 mMdithiothreitol, 30 mM-bovine serum albumin, 20 #g plasmid DNA/ml, 40 to 80 ng HeLa cell topoisomerase I/ml and variable amounts of ethidium bromide. The reaction.mixture was incubated at 30°C for 30 to 60 rain. After addition of 0.2 ml of 3 M-sodium acetate, the reaction was extracted twice with a mixture of water saturated phenol/chloroform/isoamyl alcohol (1 : l : 0.04, by vol.) that had been saturated just before use with TE buffer (50 mMTris. HCI (pH 8.0) containing l raM-sodium EDTA). The aqueous phase was transferred to a glass tube, extracted twice with ether and once with n-butanol (Mallinckrodt, spectral grade). The DNA was then precipitated with ethanol, resuspended in approximately 0.6 ml of TE buffer and dialyzed against 2 1 of TE buffer overnight with one change. To determine the superhelicity of the relaxed DNA, portions of the reaction mixture were centrifuged in a cesium chloride/ethidium bromide gradient together with 14C-labeled supercoil ColEl plasmid marker. Plasmid DNA that had been treated with topoisomerase in the absence of ethidium bromide was taken to be completely relaxed (0% relative supercoiling), since a repeat exposure of topoisomerase did not alter its electrophoretic mobility. Plasmid that had not been exposed to topoisomerase was assigned a value of 100°/o relative supercoiling. On this scale, the degree of supercoiling of each partially relaxed DNA was calculated from its position in the gradient on the assumption that the separation between nicked and closed species varies linearly with superhelical density. Samples relaxed in the presence of 0-85/~g/ml and 1.70/~g ethidium bromide/ml were characterized by relative superhelical densities of 23~o and 54~/o, respectively. (c) Preparation of unique topoisomers A 30 to 40 pg sample of relaxed DNA (superhelical density 0To or 23% relative to untreated plasmid) was layered on to a 1% (w/v) agarose (type II; Sigma Chemical Co.) gel whose dimensions were 6ram thick by 10 cm wide by 20cm long. The sample was electrophoresed in a vertical apparatus (EC Apparatus Co.) at 2 V/cm for 38 h in the presence of TPE buffer (12.12 g Tris base, 0"58 g EDTA and 5"7 g 85% phosphoric acid per liter). After electrophoresis, the gel was stained for l h in T B E buffer {10.8 g Tris base, 5-5g boric acid and 0.93g N a 2 E D T A . 2 H 2 0 per liter) containing 1 #g ethidium bromide/ml. The stained gel was examined with long wavelength ultraviolet light ()~max= 366 rim) and individual topoisomers were cut from the gel. Each topoisomer was put in a dialysis bag with 4 ml of TBE buffer, placed in a horizontal electrophoresis apparatus, and electrophoresed in TBE buffer at 2.5 V/cm for 2 to 3 h. The electroeluted DNA was centrifuged to equilibrium in cesium chloride/ethidium bromide and the closed circles were extracted with n-butanol, dialyzed against TE buffer, adjusted to 0.3 M-sodium acetate, precipitated with ethanol, resuspended in 50/~l of T E buffer, and dialyzed on a filter (Miltipore SW013) against 40ml of TE buffer. Typically, 1 to 2.5pg of a given topoisomer was recovered; contamination with adjacent topoisomers was less than 10~/o. (d) Recombination reactions with unique topoisomers Integrative and excisive recombination were carried out as described (Pollock & Nash, 1983b), except that the reaction volume was 0-04 ml and each recombination reaction contained 0-4 pg of a unique topoisomeric species of an inversion substrate. After 20 min at 25°C, the reaction was stopped by addition of 10~l of a solution containing 250 mg Ficoll/m[ (Pharmacia), 50 mg sodium dodecyl sulfate/ml, and 0.3 mg bromphenol-blue/ml. The samples were electrophoresed on a 1% agarose gel containing T P E buffer for 38 h at 2 V/cm. Chloroquine (0"2 #g/ml) was added to the gel and reservoir when the substrate was a plasmid that had been relaxed to 0~/o relative superhelical density. After electrophoresis, the gel was stained with l pg of ethidium bromide/ml dissolved in TBE buffer and photographed with short wavelength ultraviolet light (~max = 310 nm). Alternatively, the gel was stained for I h with 0-1/~g DAPI (4',6'-diamidino-2-phenylindol-dihydrochloride; Boehringer Mannheim)/ml dissolved in 5mM-Tris'HC1 (pH7.4) containing 10mM-

). SITE-SPECIFIC RECOMBINATION

23

NaCI, washed twice with water and photographed with long wavelength ultraviolet light ()~ma,= 366nm). After photography, the gel was examined under long wavelength ultraviolet light and individual DNA bands were cut from the gel. The DNA in each band was electroeluted from the gel as described in section (c), above, adjusted to 0"3 M-sodium acetate, precipitated with ethanol, and resuspended in 25 gl of TE buffer. (For gels that had been stained with DAPI, re-electrophoresis of a sample of each band verified the lack of significant cross-contamination between simple circular topoisomers of differing topological linking number. However, for gels that had been stained with ethidium bromide, exposure to short wavelength ultraviolet light irreversibly altered the mobility of each topoisomer, presumably because of formation of covalent adducts between ethidium bromide and DNA.) When recombination was carried out with substrates whose relative superhelical density was 25%, the simple circular DNA in each eluted sample was separated from contaminating knots by treatment with pancreatic DNAase and re-electrophoresis in the presence of Mg2+ as described (Pollock & Nash, 1983b). After 60 h at 2.5 V/cm, the gel was stained with ethidium bromide, dissolved in TBE buffer, and examined under short wavelength illumination. The simple circular species was cut from the gel, put into a dialysis bag, electroeluted in TBE buffer overnight, precipitated with ethanol and resuspended in 25 ~l of TE buffer. Individual topoisomeric simple circles were assayed for their content of recombinants by digestion with restriction endonuclease HhaI, electrophoresis in agarose, transfer to nitrocellulose, and hybridization with 32p-labeled pBP90A6 as described in the accompanying paper (Pollock & Nash, 1983b). We routinely analyzed the same fraction (usually 1/5) of each topoisomer sample so that recombinants were displayed in proportion to their abundance in the population. Typically, 1 to 10 ng of each topoisomer were digested; reconstruction experiments showed that 0.1 ng of recombinant plasmid could be readily detected.

4. Results (a) Recombination with partially supercoiled substrates T h r o u g h o u t this work we e m p l o y e d circular s u b s t r a t e s in which a t t a c h m e n t sites divided the circle into arcs of 5 7 0 b p and 7 8 0 0 b p . As shown in the a c c o m p a n y i n g paper, integrative recombination of the supercoiled form of this s u b s t r a t e yields only k n o t t e d recombinants. A s u b s t r a t e with even shorter separation between the a t t a c h m e n t sites would be expected to have less interwrapping and, therefore, less knotting. Indeed, a s u b s t r a t e in which attP and attB are separated by only 350 bp shows a simpler s p e c t r u m of k n o t s (Pollock & Nash, 1983b). However, all or almost all of the r e c o m b i n a n t p r o d u c t s of the s u b s t r a t e are still k n o t t e d (Nash, unpublished observation). The a m o u n t of D N A needed to constitute functional a t t a c h m e n t sites m a k e s it difficult to construct an active recombination s u b s t r a t e with a separation between cores m u c h smaller t h a n 350 bp. Therefore, in order to obtain some u n k n o t t e d r e c o m b i n a n t products, we chose to reduce the degree of supercoiling of the s u b s t r a t e whose a t t a c h m e n t sites are separated by 570 bp. We are not free to reduce supercoiling at will. The topoisomerase a c t i v i t y of I n t can relax D N A in a fashion t h a t is uncoupled from r e c o m b i n a t i o n (Kikuchi & Nash, 1979a). To meaningfully interpret changes in the topological linking number, recombination m u s t be rapid relative to relaxation. E v e n under optimised conditions, non-supercoiled D N A recombines slowly (Pollock & Nash,

24

H. A. NASH AND T. J. POLLOCK

1983a). We examined substrates of different degrees of superhelicity in order to find a compromise superhelical density high enough to permit reasonably prompt recombination but low enough to permit formation of unknotted recombinants. As shown in Figure l(a), lane 3, a substrate whose superhelical density is 23~/o that of untreated plasmid yields a substantial amount of recombinant product in 20 minutes. In parallel experiments, we have found that relaxation of supercoils by Int is minimal at this time (data not shown). The substrate whose superhelical density was reduced to zero yields barely detectable recombinants within 20 minutes (Fig. l(a), lane 4). This substrate showed more recombination after one hour but a parallel experiment showed that Int topoisomerase had caused substantial relaxation of a unique topoisomer during this time. Figure l(b) shows that both simple circles and trefoils are found in reaction mixtures using the partially supercoiled substrate. The unknotted and knotted forms were eluted from the gel. Restriction analysis of the eluted DNA demonstrated that about one-quarter to one-fifth of the recombinant product was present as simple circles; the remainder was found as trefoils (data not shown). Thus, it appears that reducing the superhelical density to 23~/o offers a reasonable compromise between the speed of recombination and the complexity of the recombinant product. With such a substrate, decreasing the time of incubation to five minutes or raising the KCl concentration of the reaction mixtures to 70mM decreases the yield of recombinant molecules drastically (data not shown). We have not investigated the behavior of substrates whose superhelical density has been reduced to values between zero and 23O/o of normal. (b) Integrative recombination with unique topoisomers Individual topoisomers were purified from a population of partially relaxed closed circular inversion substrate molecules. The individual topoisomers were reacted with purified Int and I H F for 20 minutes under conditions permissive for recombination of non-supercoiled DNA. The reaction mixtures were then electrophoresed in agarose; adjacent lanes contained a sample of unreacted topoisomer and a sample of the 23~/o supercoiled population from which the unique topoisomer had been purified. Figure 2 shows the results from a typical experiment. One notices first that extensive relaxation has not occurred. That is, after recombination most of the DNA migrates near the position of the starting topoisomer (Fig. 2(a), lanes 1 and 3). This confirms and extends the earlier conclusion that a large amount of swivelling of DNA strands is not an intrinsic part of the recombination mechanism (Mizuuchi et al., 1978,1980a). This result also shows that the uncoupled Int topoisomerase is not very active under our conditions. Closer inspection of Figure 2(a), lane 3 shows that several new bands are produced in the reaction mixtures; all of these bands depend absolutely on the presence of Int protein (data not shown). Two strong bands are resolved near the position of the substrate topoisomer (these two bands are contained in the area marked by the bracket labelled 0 in Fig. 2(a), lane 3). We reasoned that one was a simple circle with the same linking number as the substrate and the other was a knot. This was confirmed in the following way. We cut each band from the gel,

), SITE-SPECIFIC RECOMBINATION

25

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Fie. 1. Integrative recombination with partially supercoiled substrate. (a) Integrative recombination was carried out with pBP90A6 substrate as described (Pollock & Nash, 1983b}; the superhelix density of the substrate (relative to the untreated plasmid} was 100% (lane l}, 54~/o (lane 2), 23% {lane 3), or 0% {lane 4). After 20 rain, the reaction mixtures were treated with HhaI endonuclease, electrophoresed in polyacrylamide and photographed as described {Pollock & Nash, 1983b}. The position of the recombinant fragment containing attR is indicated. (b} A portion of the reaction mixture analyzed in lane 3 was treated with pancreatic DNAase and electrophoresed as described (Pollock & Nash, 1983b) to separate simple circular from knotted circular species. The nicked simple circular and trefoil species are marked by a filled and an open arrowhead, respectively.

eluted the D N A and treated portions with pancreatic D N A a s e to nick the circles; electrophoresis in agarose in the presence o f Mg 2+ then identified the topological forms in each band. The s l o w e r - m o v i n g and f a s t e r - m o v i n g bands contained, respectively, trefoil k n o t s and simple circles as the p r e d o m i n a n t species (data n o t shown). A l t h o u g h the trefoil band contained s o m e simple circles and vice versa, w e believe that this is due to c o n t a m i n a t i o n o f each band w i t h D N A from its neighbor and reflects the minimal resolution o f the simple circular and k n o t t e d species. In Figure 2(a), fainter b a n d s can be seen t h a t migrate as e x p e c t e d for

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Fro. 2. Integrative recombination with a unique topoisomer. (a) Lane 1 contains a sample of the substrate DNA. This topoisomer was prepared from the population of partially supereoiled circles that is shown in lane 2. A sample of the unique topoisomer was recombined for 20 min (lane 3). Recombination ~aixtures and eleetrophoresis conditions are described in Materials and Methods• Circular DNA that had changed linking number by 0, + 1 or + 2 topological ~urns from the substrate was cut from the gel as indicated by the brackets. Knotted DNA t h a t contaminated these circles was removed by a 2nd cycle of ~lectrophoresis as described in Materials and Methods. (b) The purified simple circles were restricted with HhaI endonuclease, electrophoresed in agarose, ~ransferred_to nitrocellulose paper and hybridized as described in Materials and Methods. The leftmost lanes show restriction patterns of pure pBP90A6 and

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SITE-SPECIFIC RECOMBINATION

27

simple circular topoisomers relaxed by one or two steps from the initial substrate. Note that the band relaxed by two steps is more intense than the others. Very faint bands can be detected in Figure 2(a) that correspond to DNA that has changed linking number by three and four steps. These bands were not present in most experiments and never contained enough DNA for further study. Additional faint bands that interleave between the simple circular species are presumably relaxed forms of the knotted circle. We determined how recombinant DNA is distributed among the simple circles in the following way. We cut out each band corresponding to a simple circle of different linking number. To ensure that all of a given topoisomer was isolated, we cut each band liberally, including a substantial amount of knotted DNA. We then removed the contaminating knots by electrophoresis in the presence of Mg 2+ after nicking the DNA with pancreatic DNAase (Liu et al., 1980). The nicked simple circles that derived from topoisomers with linking number changed by 0, ÷ l, or + 2 from the substrate were eluted from this second gel and analyzed for recombinants by restriction. As shown in Figure 2(b), recombinants are found only in circles that had changed their linking number by two. All the simple circles that had not changed linking number were simply unreacted substrate. All the simple circles that differed by a linking number of one from the initial substrate were also unreacted substrate. They presumably arose from the action of Int functioning as a relaxing enzyme uncoupled from recombination; this confirms the type I character of Int topoisomerase (Nash et al., 1981). On longer exposure of the transfer shown in Figure 2(b), a faint amount of such unreacted substrate can be seen also in the material that has changed linking number by two. This entire analysis has been repeated on two occasions with different batches of Int, I H F and substrate. In each case, the same pattern is observed: recombinants appear only in simple circles that have lost two superhelical turns. (c) Excisive recombination with unique topoisomers Recombination between prophage attachment sites differs from integrative recombination in several ways (reviewed by Echols & Guarneros, 1983). For example, excisive recombination is more thermoresistant and less dependent on supercoiling than is integrative recombination (Abremski & Gottesman, 1979). Furthermore, in addition to Int and IHF, excisive recombination requires the action of a third protein, the product of the phage xis gene. Using purified Xis protein (a gift from J. Auerbach and S. Wickner), Int and IHF, we carried out excisive recombination on an inversion substrate in which the prophage sites were separated by 570 bp. Figure 3 shows a typical result for a single topoisomer purified from a population of substrate circles. As for the study of integrative recombination, the average superhelical density of this population had been adjusted to 23~/o by relaxation in the presence of ethidium bromide. Note that the electrophoretic pattern of DNA from the excisive recombination reaction (Fig. 3(a)) is much simpler than that from the integrative recombination reaction (Fig. 2(a)). The most intense bands migrate as expected for simple circles with topological linking numbers changed by 0, + l , +2, or + 3 from the original

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FIo. 3. Exeisive recombination with a unique topoisomer. (a) The substrate DNA is shown in lane 1. It was purified from a population of partially relaxed ~ircles of pLR90A6; a sample of this population is shown in lane 2. The substrate was reeombined for 20 min with purified Int, I H F and Xis, the reaction ~ixture was then electrophoresed in lane 3. Simple circles that had changed linking number by 0, + 1, + 2 or + 3 turns relative to the substrate were cut from ;he gel as indicated. The DNA in each region was freed from contaminating knots as described in Materials and Methods. (b) The purified simple circles of tifferent linking number were digested with HhaI endonuclease, transferred to nitrocellulose, and hybridized with 32P-labeled probe as described in Materials ~nd Methods. The recombinant band that contained attP (P) and attB {B) are indicated.

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2 SITE-SPECIFIC RECOMBINATION

29

substrate topoisomer. The band corresponding to a circle that has changed linking number by two is quite intense. One or two faint bands interleave between these topoisomers; these presumably represent a small amount of knotted DNA. The relative absence of knots in excisive recombination as opposed to integrative recombination when partially supercoiled DNAs are used as substrate agrees with our earlier studies that used fully supercoiled and fully relaxed substrates (Pollock & Nash, 1983b). Individual topoisomer species generated by excisive recombination were eluted from the gel and freed from contaminating knots by treatment with pancreatic DNAase and a second electrophoresis. The purified simple circles were assayed for recombinant DNA by restriction analysis. Figure 3(b) shows that almost no recombinant is found in circles of a change in linking number with 0 or + 1. The latter represent unreacted substrate that has been relaxed by one unit by Int topoisomerase. This shows for the first time that Xis does not alter the type I character of the relaxing activity of Int (Nash et al., 1981). Most of the simple circular recombinant product is found in topoisomers that have changed linking number by +2. We believe the recombinant found in topoisomers with linking changes of + 3 reflects the action of Int topoisomerase either on the substrate or, more likely, on the recombinant product itself. Regardless of their source, recombinants with topological linking change of +3 arc not a major species. By comparing the intensity of recombinant fragment bands derived from different amounts of the various knot-free species, we conclude that at least 80% of the simple circular recombinant product is contained in a species with topological linking change of + 2. (d) Excisive recombination with a non-supercoiled topoisomer We have shown that both integrative and excisive recombination of a partially supercoiled substrate leads to a small positive change in topological linking number. The sign of this change is that favored by the energetics of supercoiling, i.e. recombination leads to a small relaxation of negatively supercoiled substrate. To test whether the direction of the change in linking number is fixed by the recombination machinery or simply responds to supercoiling, we wish to examine recombination of a topoisomer that is not under superhelical strain. Unfortunately, as shown in Figure l, integrative recombination is so slow on fully relaxed substrates that it is hard to detect recombinants at reasonably short incubation times. However, we have found that excisive inversion proceeds with adequate speed even on fully relaxed substrates. This agrees with the conclusion drawn from earlier studies on the kinetics of intramolecular integrative and excisive recombination using substrates with directly repeated attachment sites (Abremski & Gottesman, 1979; Pollock & Abremski, 1979). Figure 4(a) shows the results of recombination with a unique topoisomer purified from a population of fully relaxed excisive inversion substrate circles. All visible bands migrate as expected for simple circular topoisomers. In agreement with the results presented in the accompanying paper, no knotted forms are observed. It should be pointed out that in the gel system shown in Figure 4, slower moving bands have lower topological linking numbers. This is

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FIG. 4. Excisive recombination of a fully relaxed topoisomer. The substrate, shown in lane 1, was purified from a population of circles tl~at had been relaxed the absence of ethidium bromide; a sample of this population is shown in lane 2. The substrate was recombined and electrophoresed as for Fig. 3, except hat the gel contained 0-2/zg chloroquine/ml. Circles that had changed linking number by 0, - 1 or - 2 turns relative to the substrate were cut from the gel as adicated. (b) Circles of different linking number were analyzed for recombination by digestion with HhaI endonuclease as described for Fig. 3. The ecombinant bands that contained attP (P) and attB (B) are indicated.

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2 SITE-SPECIFIC

RECOMBINATION

31

demonstrated by the behavior of populations of circles that have been relaxed at different temperatures. Circles that are relaxed at higher temperatures and therefore have lower topological linking numbers (Depew & Wang, 1975) migrate slower in this gel system (data not shown). The difference in electrophoretic behavior in the gel of Figure 4(a) and that for the gels shown in Figures 2(a) and 3(a) reflect both the gel condition (addition of chloroquine) and the initial state of the DNA (relaxed). Individual topoisomers were eluted from the gel and analyzed for recombination by restriction. As shown in Figure4(b), the bulk of the recombinants are found in a topoisomer that had changed its topological linking number by - 2 . It therefore appears that the direction in the change of the linking number is not fixed by the recombination mechanism but responds to the energetics of supercoiling. If substrate DNA were completely relaxed, one would expect equal amounts of recombinants that have increased and decreased their topological linking number. In the example shown in Figure 4(a), it appears that the topoisomer substrate was slightly positively supercoiled under recombination conditions and therefore yielded recombinants primarily with lower values of the topological linking number. A different topoisomer substrate purified from the same relaxed population did in fact yield recombinants that had changed linking number by ÷ 2 as well as recombinants that had changed linking numbers by --2 (data not shown). 5. Discussion

(a) Recombination-provoked change in linking number:

general implications for synapsis The major result of this paper is that simple circular products of site-specific inversion differ in supercoiling from their circular parents by two topological turns. Although the precise change in linking number of knotted recombinants is not easily defined, in every case where knotted products accumulate, a single species of knot predominates. This shows that the knotted recombinants have also undergone a unique change in linking number and indicates that all lambda sitespecific recombination is accompanied by a characteristic alteration in supercoiling. This is an important result, because it places limits on acceptable models for synapsis. Synapsis is defined as the stage of recombination in which the recombining DNAs are juxtaposed. Although such juxtaposition is a logical requirement for a breakage and reunion recombination, neither genetic nor biochemical studies have shed much light on even the gross organization of this step. For example, we are ignorant as to whether synapsis occurs before or after cleavage of parental DNA strands. In addition, we do not know whether synapsis brings the parental DNAs into intimate contact or whether the two parents are simply placed in the same vicinity and permitted to find each other by diffusion in a restricted space. The finding that a unique change in linking number accompanies recombination argues strongly that synapsis is early and tight. Consider what would happen if synapsis occurred after cleavage of parental DNA. In response to random thermal

32

H. A. NASH AND T. J. POLLOCK

forces and/or the strain of supercoiling, one would expect the broken parental ends to rotate until synapsis aligned them and permitted rejoining to finish the recombination. Since the amount of time between cleavage and synapsis would not be fixed, in each molecule the amount of swiveling might be different. The circular products of recombination would thus be expected to differ from the substrate by a variable number of topological turns; the product circles would be distributed over a ladder of different topological linking numbers. The same expectation follows if synapsis occurs before strand cleavage but involves not a tight but a loose approximation of parental DNAs. Here, the broken ends would diffuse in a volume defined by the synaptic structure; during the time required for this diffusion, swiveling of the broken ends would unwind DNA and lead to recombinants with varying linking numbers. As pointed out in the Introduction, earlier studies showed that the bulk of the substrate supercoils are retained in recombinant products (Mizuuchi et al., 1978,1980a). This ruled out the most extreme models of late and/or loose synapsis. The present results sharpen this limitation decisively; for the vast majority of recombinant products, no random swiveling has occurred at all. It would be interesting to know how much time it takes for DNA to swivel around a break point. Unfortunately, there are no direct experiments that address this subject. However, Davison (1966) has shown that complete unwinding of the DNA of bacteriophage T2, containing some 16,000 turns, occurs in less than 25 seconds. In addition, the kinetics of branch migration, a process that involves swiveling as well as other internal motions of DNA, can occur at speeds consistent with a calculated rate of l0 s events per second (Radding et al., 1977). Clearly, the synapsis step during site-specific recombination either never lets go of the broken DNA or, at most, permits it no more than a few microseconds of freedom. (b) Specific proposals for synapsis: four-stranded DNA Kikuchi & Nash (1979a) have proposed a detailed model for site-specific recombination that suggests how tight synapsis and very limited motion of DNA strands might be achieved. In this model, synapsis of attachment sites occurs by the formation of a four-stranded helix involving the homologous core of attP and attB (Fig. 5(a)). Kikuchi & Nash postulated that cleavage of parental strands takes place at the margins of the four-stranded region (Fig. 5(c)) and is carried out by the topoisomerase function of Int. At each margin, one strand from each parent is cut; strand exchange occurs when each parental double helix swivels around the uncut strand. As a result of the very close juxtaposition of parental DNAs in the proposed four-stranded synapsis, swiveling is limited to a fraction of a turn, since broken DNA immediately encounters a broken end from the other parent and is promptly rejoined. As shown in Figure 5(b), if synapsis utilizes the kind of four-stranded DNA structure first proposed by MeGavin (1971), each parental DNA chain joins the four-stranded region at one corner of a square. Clockwise rotation around the uncut strand of 270 ° then brings a broken end into apposition with a broken end from the other parent (Fig. 5(d)). How does the experimentally determined change in linking number compare

S I T E - S P E C I F I C RECOMBINATION

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lOOVgIIIIi II l II Fro. 5. Strand exchange mechanism with 4-stranded DNA synapsis. (a) Two double helices synapsed in a 4-stranded helix of the kind proposed by McGavin (1971). (b) The stereochemistry of the 4-stranded regions as seen in a cross-section perpendicular to the helix axis. The bases from each of the 2 parents are marked with heavy and light lines, respectively. The arrowheads indicate the strand whose 5' end is above the plane of the page. Note the non-Watson-Crick hydrogen bonds that connect the 2 double helices; these hydrogen bonds (and similar ones formed with G- C pairs) serve to stabilize 4-strand helices made from homologous sequences (McGavin, 1971). (c) The same structure as in (a) but with all the helical character removed for simplicity. The knobs at the end of each strand indicate the 5' end. Cleavage of DNA by Int at the border of the 4-stranded regions, indicated by arrows, initiates recombination. In (d), the rotations required to bring the cut strands adjacent to new partners are indicated. (e) The result of resealing of the newly aligned strands; a Holliday structure is formed. Recombination can be completed by repeating the cycle of cleavage, rotation, and resealing on the remaining pair of strands. (f) to (J) The same mechanism with the minor groove pairing scheme of Wilson (1979) used to form 4-stranded synapsis. Note that in (g) the Watson-Crick hydrogen bonds connects bases from different parents.

w i t h t h a t p r e d i c t e d from t h e a b o v e model? N a s h et al, (1981) r e p o r t e d t h a t u n i o n of t w o circles b y this m e c h a n i s m is c a l c u l a t e d to y i e l d a p r o d u c t circle whose l i n k i n g n u m b e r is c h a n g e d b y four from t h a t of its s u b s t r a t e s . This c h a n g e is t h e s u m of t h r e e topological t u r n s d e r i v e d from t h e s w i v e l i n g of p a r e n t a l s t r a n d s (4 x 2700/360 °) plus one t o p o l o g i c a l t u r n f r o m a c h a n g e in w r i t h i n g as a figure 8 i n t e r m e d i a t e resolves to a r e c o m b i n a n t (see M c G a v i n (1983) for f u r t h e r d i s c u s s i o n of this p o i n t ) . I n c a l c u l a t i n g t h e l i n k i n g c h a n g e for i n t r a m o l e c u l a r i n v e r s i o n , t h e

34

H. A. NASH AND T. J. POLLOCK

same considerations apply, except that the writhing term is of opposite sign to the twisting term so that the predicted change in linking number is % 3 - 1 = ÷ 2 topological turns. This is, of course, precisely the change in linking number found in our experiments. It should be pointed out that several non-trivial variants of the scheme described above will yield a similar predicted linking number change. For example, broken strands can also reach new partners by a 90 ° rotation (Fig. 5(d)). For an intramolecular inversion, such motion yields recombinants that have also changed linking number by two. However, because the 90 ° rotation is in the opposite direction to that for a 270 ° rotation, the change in linking number is (4 x --90°/360 ° ) - 1 = --2 topological turns. Kikuchi & Nash (1979b) assumed that 90 ° rotations were forbidden because the rotating double helices would bump into one another. (By contrast, rotation in the opposite direction swings each double helix out away from its recombining partner.) If the four-stranded DNA border is more flexible or less tightly packed than originally presumed, 90 ° rotations might be allowed. This possibility becomes more interesting when one considers a second variation in four-stranded DNA models for site-specific recombination. Wilson (1979) has proposed that four-stranded DNAs with the identical stereo-chemical structure as that proposed by McGavin can be derived by an altogether different pathway. Rather than wrap two DNA molecules around each other's major groove, as proposed by McGavin, Wilson (1979) pointed out that each double helix could pair along the minor groove, then melt and reanneal with its partner to form a four-stranded DNA that consists of two Watson-Crick heteroduplexes wrapped about each other (Fig. 5(f) and (g)). Nash et al. (1981) pointed out that a similar scheme of cleavage of strands and partial rotation can be carried out on this putative synaptic intermediate to yield recombinants. However, the sense of rotation must be reversed; thus, the 270 ° rotations are counter-clockwise (Fig. 2(i)) and, for an inversion, yield recombinants with a linking change of - 2 . However, if 90 ° rotations are sterically permitted, synapsis following the Wilson pathway could also produce recombinants with linking change of ÷ 2. In summary, the observed changes in linking number can be accounted for easily by the hypothesis that DNA synapsis occurs via a four-stranded helix and that strand exchange involves cleavage of DNA at the edges of this structure followed by rotation of the parental DNA. The measurement of changes in supercoiling does not permit us to distinguish between several variants of this hypothesis. In the next section, we consider hypothetical mechanisms for recombination that can be rejected on topological grounds. (c) Models for recombination that predict zero linking change McGavin (1977) pointed out that simultaneous cleavage of all the strands in a four-stranded helix would permit rotation of one segment of the structure with respect to the other. After a rotation of 180°, the broken ends of DNA would be opposite new partners and ligation would yield a pair of reciprocal recombinants. However, for an inversion substrate, this mechanism would produce recombinants

), SITE-SPECIFIC RECOMBINATION

35

whose linking number was identical to that of the substrate; it is therefore not an acceptable mechanism for lambda site-specific recombination. Sherratt et al. (1981) suggested an altogether different model for synapsis during site specific recombination. They proposed that parental DNAs are brought into intimate contact as a result of proteins bound to the recombining sitvs. In their model, a four-stranded helix is not formed but the two DNAs are held near each other as a result of protein-protein interaction. The DNAs are thought to be arranged so that they lie crosswise on top of one another. After strand cleavage via a topoisomerase, only a small shift in position permits the broken strands to find new partners. Although a particular pathway for this strand shifting is not spelled out in the original proposal, studies with models (Nash, unpublished results) indicate that the simplest strand motion yields recombinant products from an inversion substrate that have not suffered a change in linking number. On this basis, the model proposed by Sherratt et al. (1981) is also ruled out as a mechanism for lambda site-specific recombination. Of course, it may be that the recombination proteins direct a movement of broken strands that is more complex than what is easily achieved with rubber tubing; depending on the strand motion hypothesized, recombinants with altered linking number could result from the Sherratt mechanism. (d) Four-stranded D N A and the role of homology in recombination Because of the paucity of biochemical data on the actual structures involved in synapsis, it should be possible to construct many models that would agree with our topological findings. However, there is an additional reason to prefer the class of model that invokes a four-stranded helix as a synaptic intermediate: these models easily account for the role of homology in lambda site-specific recombination. As pointed out in the original proposal for the stereochemistry for the four-stranded DNA (McGavin, 1971), the structure is stabilized by hydrogen bonds that form between the two parental DNAs if they share homology (Fig. 5(b)). Heterologous base-pairs do not fit into a regular four-stranded structure and/or lack the capacity for these hydrogen bonds. Lambda site-specific recombination takes place within a 15 base-pair homologous sequence (Landy & Ross, 1977). Recent studies on attachment site variants have shown that some of this sequence can be altered in one attachment site with little or no change in recombination efficiency in vivo if, and only if, the identical sequence alteration is made in the partner site (Mizuuchi et al., 1981; Weisberg et al., 1983). This demonstrates that the lambda recombination mechanism senses homology. However, these experiments do not tell us which stage in recombination requires homology. Weisberg et al. (]983) have suggested that, as an alternative to a putative role for homology in synapsis, homology in the attachment site core may be required only after one pair of strands has exchanged. In this view, branch migration through the homologous region would be required to move the point of strand exchange to a new position, where a second cluster of recombination proteins could carry out the exchange of the remaining pair of strands. We think that this is an unlikely hypothesis for the following reason. Site-specific

3{i

H.A. NASH AND T. J. POLLOCK

recombination in vitro has been attempted within an attB site that contains mutant bases in the core region (Nash, unpublished results). When paired with a wild type attP site, no novel DNA species could be detected after incubation with Int and IHF. If homology were important only for branch migration, we would expect to accumulate intermediates that contain Holiday junctions, i.e. that had exchanged only one pair of strands. No such structures were seen, even though restriction digestion of the reaction mixtures would have produced a species of predictable mobility that could have been detected if it represented conversion of 5% of the substrate. However, in agreement with the studies in vivo reported by Weisberg and his collaborators (Mizuuchi et al., 1981; Weisberg et al., 1983), when an altered attB site was reacted with an attP carrying the same alteration integrative recombination in vitro proceeded with the same efficiency and kinetics (30% of the substrate recombined in 30 min) as did recombination of wild type sites. Since these data suggest that homology is not required solely for branch migration, we prefer to believe that homology is important for an earlier step in recombination. Models that invoke four-stranded DNA synapsis provide a ready explanation for this feature. (e) The relationship between integrative and excisive recombination Since the discovery of the xis gene, it has been understood that integration and excision are not simply the same reaction run in either of two directions. However, it has been possible to imagine that the simple reversal of integration was kinetically blocked and that the function of Xis was to release this block and permit the integration reaction to run backwards (Nash, 1981). If that were the case, then the change in topological linking number for excision should be precisely the opposite of that for integration. Our observations rule out the strictest version of this hypothesis. With substrates that are partially supercoiled, both integrative recombination and excisive recombination yield recombinant products that have undergone the same change in linking number; a relaxation of two topological turns. This result tempts one to favor an alternative hypothesis: that Xis permits the prophage sites to use precisely the same pathway as integration. However, our observation that, when supercoiling is removed as a driving force, excisive recombination can yield either a positive or negative change in linking number limits our ability to make a strong deduction about the mechanism. This is illustrated by considering alternative mechanisms for excisive recombination, all of which invoke the formation of four-stranded DNA as a synaptic intermediate. First, suppose that at the borders of the four-stranded helix, 90 ° rotation of broken strands is forbidden for steric reasons. Then, in order to explain our observation that excisive recombination can change linking numbers either by + 2 or - 2 , we must conclude that synapsis can take place either by the McGavin pathway, where 270 ° clockwise rotation changes the linking number by + 2, or by the Wilson pathway, where 270 ° counterclockwise rotation yields recombinants with linking numbers changed by - 2 . In this case, there is no single pathway for excisive recombination and it becomes meaningless to ask whether integration and

2 SITE-SPECIFIC RECOMBINATION

37

excision always use the same pathway. On the other hand, if the ibur-stranded DNA is flexible enough to permit 90 ° rotation of broken ends to realign parental DNAs, recombinants with a + 2 or --2 change in linking n u m b e r can be generated from either the McGavin or Wilson p a t h w a y (see above). In this case, we cannot tell whether excisive recombination always uses the same p a t h w a y as integrative recombination, always uses the opposite pathway, or chooses either p a t h w a y at random. Thus, experiments on linking n u m b e r changes do not sharply distinguish between different modes of synapsis for excisive recombination versus those of integrative recombination. This uncertainty also limits our capacity to distinguish between alternative explanations for the frequency of knotting during integrative and excisive recombination (Pollock & Nash, 1983b). The resolution of these questions must await more direct biochemical experiments. We thank J. Auerbach and S. Wickner for a gift of Xis protein and L. Liu for a gift of HeLa topoisomerase I. We are grateful to S. McGavin for discussions of the topological consequences of recombination. N. Craig, P. Kitts and R. Weisberg are thanked for their comment on this manuscript, which was ably prepared by L. Burkhardt. REFERENCES Abremski, K. & Gottesman, S. (1979). J. Mol. Biol. 131,637-649. Campbell, A. M. (1981). Cold Spring Harbor Symp. Quant. Biol. 45, 1-9. Davison, P. F. (1966). J. Mol. Biol. 22, 97-108. Depew, R. E. & Wang, J. C. (1975). Proc. Nat. Acad. Sci., U.S.A. 72, 4275-4279. Echols, H. & Guarneros, G. (1983). In The Bacteriophage Lambda I I (Hendrix, R.W., Roberts, J. W., Stahl, F. W. & Weisberg, R. A., eds), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, in the press. Kikuchi, Y. & Nash, H. A. (1979a). Proc. Nat. Acad. Sci., U.S.A. 76, 3760-3764. Kikuchi, Y. & Nash, H. (1979b). Cold Spring Harbor Syrup. Quant. Biol. 43, 1099-1109. Landy, A. & Ross, W. (1977). Science, 197, 1147-1160. Liu, L. F., Liu, C.-C. & Alberts, B. M. (1980). Cell, 19, 697-707. McGavin, S. (1971). J. Mol. Biol. 55, 293-298. McGavin, S. (1977). Heredity, 39, 15-25. McGavin, S. (1983). J. Theoret. Biol. In the press. Mizuuchi, K., Gellert, M. & Nash, H. A. (1978). J. Mol. Biol. 121,375-392. Mizuuchi, K., Gellert, M., Weisberg, R. A. & Nash, H. A. (1980a). J. Mol. Biol. 141,485494. Mizuuchi, K., Fisher, L. M., O'Dea, M. H. & Gellert, M. (1980b). Proc. Nat. Acad. Sci., U.S.A. 77, 1847-1851. Mizuuchi, K., Weisberg, R., Enquist, L., Mizuuchi, M., Buraczynska, M., Foeller, C., Hsu, P.-L., Ross, W. & Landy, A. (1981). Cold Spring Harbor Syrup. Quant. Biol. 45, 429-437. Nash, H. A. (1981). Annu. Rev. Genet. 15, 143-167. Nash. H. A. & Robertson, C. A. (1981). J. Biol. Chem. 256, 9246-9253. Nash, H. A., Mizuuchi, K., Enquist, L. W. & Weisberg, R. A. (1981). Cold Spring Harbor Syrup. Quant. Biol. 45, 417-428. Pollock. T. J. & Abremski, K. (1979). J. Mol. Biol. 131,651-654. Pollock, T. J. & Nash, H. A. (1983a). In Genetic Rearrangement (Chater, K. F., Cullis, C. A., Hopwood, D. A., Johnston, A. W. B. & Woolhouse, H. W., eds), pp. 41-58, Croom Helm, London. Pollock, T. J. & Nash, H. A. (1983b). J. Mol. Biol. 170, 1-18. Radding, C. M., Beattie, K. L., Holloman, W. K. & Wiegand, R. C. (1977). J. Mol. Biol. 116, 825-839.

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Sherratt, D., Arthur, A. & Dyson, P. (1981). Nature (London), 294, 608-610. Shure, M. & Vinograd, J. (1976). Cell, 8, 215-226. Weisberg, R. A. & Landy, A. (1983). In The Bacteriophaqe Lambda I I (Hendrix, R. W., Roberts, J. W., Stahl, F. W. & Weisberg, R. A., eds), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, in the press. Weisberg, R,. A., Gottesman, S. & Gottesman, M. E. (1977). Compr. Virol. 8, 197-258. Weisberg, R. A., Enquist, L. W., Foeller, C. & Landy, A. (1983). J. Mol. Biol. In the press. Wilson, J. H. (1979). Proc. Nat. Acad. Sci., U.S.A. 76, 3641-3645. Edited by M. Gellert