Strand exchange in site-specific recombination (phage X/Holliday model)

Transcription

1 Proc. Nati. Acad. Sci. USA Vol. 76, No. 3, pp , March 1979 Genetics Strand exchange in site-specific recombination (phage X/Holliday model) L. W. ENQUST*t, H. NASHf, AND R. A. WESBERG* *Laboratory of Molecular Genetics, National nstitute of Child Health and Human Development, National nstitutes of Health, Bethesda, Maryland 214; and tlaboratory of Neurochemistry, National nstitute of Mental Health, Bethesda, Maryland 214 Communicated by M. S. Meselson, December 11, 1978 ABSTRACT The site-specific recombination system of phage A promotes crossovers at its attachment site (att). n this report we show that when phage are crossed in conditions where only the site-specific recombination system is active, a low frequency of crossovers can also be detected in a region that is close to but does not contain att. These crossovers require the phage int gene, the host hip gene, and the integrity of att. They are not detected if one of the parents carries a substitution of a heterologous attachment site (attb instead of attp) To explain these findings we suggest that site-specific recombination can proceed by exchange of single strands between the participating chromosomes at att and migration of the resulting junction outside of att. The int gene product of phage A promotes recombination between DNA molecules carrying specific nucleotide sequences called attachment sites. The role of int-promoted recombination in the life cycle of X is to insert the phage chromosome into and to excise it from the host chromosome (see refs. 1-3). Previous work has shown that crossing over promoted by int gene product proceeds by breakage, exchange, and rejoining of the four participating polynucleotide strands (see ref. 4). We would like to consider two pathways of strand exchange: concerted, in which the four strands-are exchanged simultaneously, and independent, in which the strands are exchanged one or two at a time. A model of concerted strand exchange (5, 6) is described in Fig. 1, structures A and B. n this model, an enzyme introduces staggered single-strand breaks at the ends of a short segment in each of the two participating attachment sites. Strand breakage is followed by melting, reciprocal exchange, and resealing. The sequences that are cut and reannealed must be identical in the two attachment sites. ndeed, nucleotide sequence analysis has shown that the bacterial and phage attachment sites do possess a common sequence 15 nucleotide pairs long (the core sequence) (7). A model for independent strand exchange is depicted in Fig. 1, structures C through G. Breakage and reciprocal exchange of two strands of the same chemical polarity, which we assume occurs within the core sequence, will generate structure D, a crossed-strand exchange or Holliday structure. This structure was proposed as a recombinational intermediate by Holliday (13) and was shown to be structurally reasonable by Sigal and Alberts (14). t has been observed in electron microscope studies of intracellular phage and plasmid DNA (see refs. 8 and 15 and references cited therein). The site of strand exchange in a Holliday structure can migrate with respect to a fixed point on the molecule (Fig. 1, D and E) (9-11). n order to produce genetic recombinants, the Holliday structure must be resolved to give two unconnected duplexes. This can occur by cutting, exchange, and resealing of two strands of the same chemical The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C solely to indicate this fact polarity as described in the legend to Fig. 1. The two possible genetic outcomes are structure F (outside markers recombined) and structure G (outside markers parental). f int-promoted recombination proceeds exclusively by concerted strand exchange, then crossovers should occur only in intervals that contain an attachment site. n contrast, if intpromoted recombination can proceed by independent strand exchange, then migration of the resulting Holliday structure will sometimes lead to crossing over at a point that is distant from the initial strand exchange. The results reported in this article show that the crossovers predicted by the model of independent strand exchange do in fact occur. H. Echols (personal communication) has made similar observations. MATERALS AND METHODS Bacterial and Phage Strains. Bacterial and phage strains are listed in Table 1. Media. Media are described in refs. 16 and 17. Phage Crosses. Phage lines were crossed as described in ref. 16 except that CaCl2 (1 mm) or NaCl (.5 M) was added to the lysates to prevent loss of h phage by readsorption to debris (M. Pearson, personal communication). At least 2 recombinant plaques were counted for each cross. Scoring int and xis Alleles. These markers were scored by the red plaque test (17), using one of the first five bacterial strains of Table 1 as host. We distinguished among X int +, X intam29, and X carrying int missense mutations by their different plaque types on lawns of the supf host LE423. A int + plaques have many red galactose-utilizing papillae in their centers, A intam29 plaques have fewer, and X int-missense plaques have none. RESULTS int-promoted recombination is not detectable in intervals located more than about 8 base pairs away from att in crosses between homologous parents (22, 23). We therefore assumed that int-promoted crossed-strand exchanges, if they occur, remain localized to the region near att. The region containing the int and xis genes extends rightwards about 13 base pairs from att and it is now convenient to use mutations in these genes as genetic markers (16, 17). To detect int-promoted crossovers in an interval not containing att, we crossed phages that differed in one or more int-xis region markers and also in two outside markers: h, located about 9, nucleotide pairs to the left of att, and imm, located about 8, nucleotide pairs to the right of att. Homologous recombination pathways were inactivated by mutation. The results of several such crosses are shown in Table 2, crosses 1-4. We found that about.3% of the progeny had recombined in the interval between the int marker and imm, t Present address: Laboratory of Molecular Virology, National Cancer nstitute, National nstitutes of Health, Bethesda, MD 214.

2 1364 Genetics: Enquist et al. Concerted x A -,. C< - < B x i Y Y.x Vi F x e E x, Y X s V ndependent es_. :~~~~~~~~~~~~~~1 Z X s Y _ x Y x y FG. 1. Models for site-specific recombination. The solid and open bars represent single strands of duplex DNA, the arrows on the bars give the chemical polarity of the sugar-phosphate linkage, and the letters X, x and Y, y symbolize two pairs of alleles at two genetic loci. The pathway on the left shows concerted strand exchange via a mechanism proposed by Kaiser and Wu (5) and Signer and Weil (6). n this model an enzyme introduces a pair of staggered single-strand breaks at homologous sites within the core region of each att (A). Recombination occurs by breakage of interstrand hydrogen bonds between the breaks, reciprocal double-strand exchange, reformation of the hydrogen bonds, and ligation of the nicks to yield structures B. The pathway on the right shows independent strand exchange via a Holliday structure. n this model an enzyme cuts one strand at a homologous location in each duplex (C). We assume that this point lies within the core region. The two cut strands must have the same chemical polarity. Reciprocal strand exchange followed by ligation gives the Holliday structure, D. The location of the crossover point can migrate along the duplex to give structure E (8-11). The two duplexes can be resolved from each other in either of two ways: (i) cutting of the two outside strands at the position of the vertical broken lines, 18 rotation of the arms to the right of the cuts around a horizontal axis in the plane of the page, and ligation of cuts to give structures F; or (ii) cutting of two inside strands at the horizontal broken line and ligation of the cuts to give structure G. An alternative (but less easily pictured) way of going from structure E to structures F and G, a process called isomerization, is described by Sobell (12). a region that does not contain att. We shall henceforth call this interval region. The interval between h and the int-xis marker contains the attachment site and is called region. Region crossovers represented.2-.6% of the total crossovers between h and imm. Their frequency was largely independent of the presence of nonhomology from the core region of att leftwards (cross 5). n this cross one of the parents carried the attachment site (attl) located at the left end of an integrated prophage; this site differs from attp in that phage DNA adjacent to the left end of the core is replaced by bacterial DNA (see ref. 4). Crosses carried out in the absence of functional int product showed that region crossovers were int-dependent (crosses 6 and 7). We conclude that int can promote crossovers outside of att. Site-specific recombination of phage X and of other temperate phages requires the activity of a protein encoded by a - Proc. Nati. Acad. Sci. USA 76 (1979) Table 1. Bacterial and phage strains Strain Relevant genotype Source or ref. Bacteria LE292 Hfr H arge amber (Xint-F) (16) AgalT LE394 LE292 lamb This work LE397 LE394 (Xh8O ins8) This work LE423 LE397 (k8 psupf am2) This work LE141 RW842 (Xh8 ins8) From RW842 (17) LE43 reca recb B. Egan LE427 reca stra supe QR9; (18) 594 stra supo J. Weigle; (19) 594recA stra supo reca S. Adhya N99 supo W312 N1 N99 reca MM strain 152 NK613 hip 157 reca recb A. Kikuchi QR48 reca supe (2) Phage Y513 h red3 cts857 H. Nash Y526 h intam29 red3 cts857 H. Nash Y884 h attp24 xisam6 red3 clts857 (21) Y885 h xisam6 red3 clts857 (21) G54 int2l red3 imm434 This work G541 intam2271 red3 imm434 This work G542 intam2111 red3 imm434 This work G548 ga149 int2l red3 imm434 This work G549 ga149 bio239 red3 imm434 This work G55 ga149 int6 red3 imm434 This work G551 ga149 xisl red3 imm434 This work host gene called hip. General recombination is hip independent (ref. 24; A. Kikuchi and R. Weisberg, unpublished data). s hip required for int-promoted recombination in a region not containing the attachment site? ndeed, we found that all intpromoted recombination was reduced to the level seen in int - Table 2. int -promoted recombination in regions and Parent 1 Parent 2 % recombination Cross att int att int Region Region 1 P + P P + P am P + P am P + P P + L P am29 P 21.4 <.1 7 P am29 L 21.1 <.3 8 P24 + L P + L P am29 L Parent 1 Parent 2 h att int i il i + att int immx imm 434 hregion - ' Region -* Selected markers: h and imm434. Total phage in the burst were titered on LE292. Region and region recombinants were recognized as colorless and red plaques, respectively, on LE397 (17). Percent recombination is (number of recombinants. total phage) X 2. For crosses 1-3, the host was N1; for crosses 4-7, the host was 594recA. n crosses 1-5 and 1, parent 1 was Y526; in crosses 6 and 7, Y513; in cross 8, Y884; and in cross 9, Y885. n crosses 1, 4, and 6, parent 2 was G54; in crosses 5, 7, 8, 9, and 1, G548; in cross 2, G541; and in cross 3, G542. We estimate the int2l mutation is located within 1 base pairs of att, while intam2271 and intam2111 lie just to the right of int2l and are within 2 base pairs of att (16). i

3 Table 3. Genetics: Enquist et al. Effect of host and heterology on recombination int of att of % recombination Cross hip parent 1 parent 2 Region Region P P L L B am29 B.5.2 The design of all crosses was as illustrated in Table 2, and progeny phage were assayed as described in the legend to Table 2. For crosses 5 and 6, region was defined as extending from the right end of the attb substitution, which lies within int, to the left end of the imm434 substitution. Crossovers within this region should generate X h int + imm434 progeny, while crossovers within the core region should generate A h int-deletion imm434 progeny. Parent 1 was Y513 in crosses 1-5 and Y526 in crosses 6 and 7. Parent 2 was G54 in crosses 1 and 2, G548 in crosses 3 and 4, and G549 in crosses 5-7. The hosts were LE43 (crosses 1 and 3), NK613 (crosses 2 and 4), and 594recA (crosses 5 and 6). Cross 6 was repeated in a reca+ host (strain 594) in order to show that region recombinants can be detected when they are formed. We found 1.5% region and 1.1% region recombinants in this cross. conditions when int + phage were crossed in a hip- host (Table 3, crosses 1-4). Therefore hip is required for the production of both region and region recombinants, as expected if the two types of recombinants have a common precursor whose formation requires hip. An alternative explanation of the region crossovers is that they result from concerted strand exchange at putative secondary att sites in the int-xis region. Such sites, which are known to exist in the bacterial and phage chromosomes, participate with low efficiency in int-promoted recombination (25). This hypothesis, however, is inconsistent with the results of the following experiment. We crossed a phage carrying the attp24 point mutation with a phage carrying wild-type attl. The attp24 mutation, which is located in the core region, is known to reduce attachment site function (21) and may be a deletion of a single base pair (W. Ross, M. Shulman, and A. Landy, personal communication). We found that this mutation reduced recombination in both region and region to at least 1/2th (Table 2, crosses 8-1) and therefore conclude that little or no region recombination occurs by concerted exchange at secondary att sites. f region crossovers result from an independent strand exchange that initially occurred at att, we expect that their number will decrease as the distance of the int-xis marker from att increases. ndeed, in two separate series of experiments, we found that the frequency of region crossovers decreased to 1/8th to 1/lOth as the distance between the int-xis marker and att increased from about 1 to 13 base pairs (Fig. 2). f int-promoted recombination involves formation of a crossed-strand exchange at the attachment site core, migration into region should be blocked by any intervening nonhomology. To test this prediction, we measured the frequency of region recombination in a cross in which one of the parents carried the bacterial attachment site, attb, substituted for attp. This substitution deletes not only attp but also a small amount-about 2 nucleotide pairs (16)-of the int gene. int-promoted recombination between attp and attb can occur within the 15 base pair core sequence that is embedded in a heterologous region (7). We found that while the frequency of region crossovers was high, the frequency of region crossovers was very low and essentially independent of int function (Table 3, crosses 5 and 6). The failure to find int-promoted region recombinants in an attp X attb cross is thus consistent CD s3. Proc. Natl. Acad. Sci. USA 76 (1979) 1365 int2l int6 Kilobase pairs from att FG. 2 Frequency of recombinants as a function of distance from att. The location of the three int-xis markers was determined from the data of ref. 16, with the range of uncertainty indicated by the bars. Each of the three crosses, which were repeated twice, was of the form indicated in Table 2, with parent 2 carrying attl. Parent 1 was Y513. Parent 2 was G548, G55, or G551. The mean of the six values thus obtained for recombination in region is the -kilobase point; the range was 1.3 to 2%. The other points are region recombination frequencies. We also performed a second series of crosses, which differed from those shown by the presence of the intam29 mutation in parent 1 (Y526) and by the use of sup+ hosts for the cross and the indicator. The frequency of recombination in these crosses decreased with increasing distance from att as in the first series except that the absolute values were 2-6% lower. with the hypothesis of formation and migration of crossedstrand exchanges. However, this experiment does not show that nonhomology per se blocks migration (see Discussion). We noted earlier (see introduction) that resolution of a Holliday structure can leave outside markers either in a parental or in a recombinant configuration (Fig. 1). n contrast, region recombinants with the parental configuration of outside markers cannot arise by a single concerted strand exchange. To see if such recombinants are produced by the site-specific recombination pathway, we selected region recombinants without regard to the left-hand outside markers and subsequently scored the inheritance of these markers (Table 4). We reduced the overall frequency of recombination by using a host in which suppression of the intam29 mutation, the only source of active int protein, was poor so that only single recombination events were likely. We found (Table 4, line 1) that region recombinants with the parental configuration of outside markers were indeed produced and their frequency was 1/2 that of the nonparental type. n a second cross (line 2), in which parent 1 was int + instead of intam29, the ratio of the two recombinants was again 1:2, although the frequency of region recombinants was higher. t therefore appears that parental type region recombinants can be produced in a single intdependent event, in agreement with the model of independent strand exchange and in contradiction to the model of concerted strand exchange. The observed ratio of the two recombinant types was not predicted and we shall consider it further in the Discussion.

4 1366 Genetics: Enquist et al. Migration of a crossed-strand exchange over a region in which the two parents differ slightly will produce duplex DNA with mismatched base pairs, and these mismatches may be subject to repair (see ref. 27). Because repair can distort normal genetic linkage, an abnormally high frequency of crossovers between close markers is an argument for the existence of mismatches. We looked for and found evidence for such linkage distortion in the following cross between phage that differed by two int-xis region markers: h att intam29 +i Y513 G551 a b , ~~ + L att + xisl imm The cross was made in 594recA and the inheritance of the intam29 allele was determined among the h-imm recombinants as described in Materials and Methods. n the diagram above, recombinant a requires one crossover while recombinant b requires three. We observed that 67% of the region recombinants were of type a and 33% were of type b. A calculation based on the observed relation between recombination frequency and distance from att (Fig. 2) and assuming that multiple crossovers arise by repeated independent events predicts that the frequency of recombinant a should be 13 to 14 times that of recombinant b. DSCUSSON Our major finding is that the X site-specific recombination system promotes a low frequency of crossing over outside of but close to the attachment site. We propose that these crossovers arise from independent strand exchanges that are initiated at att and then migrate outside (see Fig. 1). Before detailed aspects of this proposal are discussed, two alternative mechanisms for the production of the observed recombinants should be considered. One mechanism invokes strand exchange that is both initiated and terminated peripheral to att-i.e., int-promoted recombination at putative attachment-like sites that are scattered over the X genome (25). This mechanism is incompatible with our observation that no int-promoted crossing over within the int gene is observed in crosses involving the attp24 mutation or attb, because in these crosses all or almost all of the int gene, and therefore the putative secondary attachment sites, remained intact. A second alternative mechanism involves the interaction of recombination enzymes with att sequences followed by movement of these enzymes away from att to peripheral sites where they promote a concerted strand exchange. Simply stated, this proposal invokes enzyme migration rather than migration of the strand exchange. Within the framework of this model, the effect of the substitution of attachment site DNA by nonhomologous DNA described above could be explained as a result of a change in path lengths for the enzyme along the DNA. The particular biotin substitution variant that we employed replaces about 2 base pairs of X DNA with over 3 base pairs of bacterial DNA. Although other substitution variants must be examined to decisively test this alternative, we do not favor it because it fails to explain our observation of Table 4. Proc. Natl. Acad. Sci. USA 76 (1979) Configuration of outside markers in region recombinants int of % % recombination parent recombination in region, X 13 1 in region a b a/b intam h Parent 1 -H attp H + or + intam29 immx _ b. Parent 2 z z ga149 int2l attl + + imm434 H Region -* Region 11H The cross was between phages Y513 and G548 in line 1 and between phages Y526 and G548 in line 2. The host was LE427, which carries the supe amber suppressor, whose activity is reduced by the presence of a restrictive stra marker (26). n order to distinguish X h imm434 progeny from X h+ ga149 imm434 progeny, the lysate was fractionated in an equilibrium CsCl density gradient. These two phage differ in DNA content by 1% and hence form well-separated peaks in such gradients. The genotype of the phage in each peak was verified by scoring imm, h, and galactose transduction (data not shown). Phage were scored for inheritance of the int marker by plating on LE397 (scores only h imm434 phage) and LE141 (scores both h and h+ imm434 phage). Recombinants of type a were recognized as red plaques on LE141 or LE397 from fractions with the density of X imm434. Recombinants of type b were recognized as red plaques on LE141 from fractions with the density of X ga149 imm434 (parent 2). Several hundred plaques of each type were scored in both crosses. Percent recombination is (number of phage in recombinant peaks X 1) - (total number of phage in the gradient). The frequencies of the two reciprocal type recombinants-x h int2l imm434 and X h+ ga149 int+/intam29 immx-were determined by scoring immunity and density and were found to be equal. An int - control (the same cross as line 1 but done in the supo host N1) gave.2% region recombinants and <.6 region recombinants. This lysate was not analyzed in CsCl gradients. frequent double and triple exchanges without further ad hoc assumptions. n contrast, initiation of strand exchange at the attachment site followed by branch migration of the strand exchange prior to its resolution produces a simple explanation for many of the phenomena we have observed. The ability of a substitution to abolish crossing over outside the attachment site can be interpreted as a block to branch migration because of the nonhomology introduced by the substitution. Furthermore, double and triple crossovers are expected consequences of the DNA heteroduplex that would be produced by branch migration. n summary, the proposal of independent strand exchange offers the simplest explanation of our data. t can also account for the formation of heteroduplex DNA within the core region, an event postulated by Shulman and Gottesman (21) to explain the segregation pattern of core mutations during site-specific recombination. t should be pointed out that although we have pictured only reciprocal strand exchange (Fig. 1), the initial strand exchange may either be reciprocal or involve transfer of only one strand; our results do not distinguish between these two possibilities. We note, however, that a nonreciprocal exchange is thought to be capable of rearrangement to yield a two-strand exchange (28).

5 Genetics: Enquist et al. The frequency of int-promoted crossovers occurring to the right of a marker located about 1 base pairs from att is only.2-.6% of the total crossovers. This low frequency can be explained in two ways. int-promoted recombination might frequently occur without the formation of crossed-strand exchanges. Alternatively, most crossed-strand exchanges might be resolved before they reach region. As shown in the following discussion, we cannot now decide between these alternatives. t has been proposed that the migration of a Holliday structure is analogous to a random walk along the chromosome (1, 11). How can this model be applied to crossed-strand exchanges in site-specific recombination? Let us assume that the random walk begins within the core region of att and that the probability of a step leftward is equal to that of a step rightward. Then, in the absence of resolution, the probability of finding a strand exchange in a given interval of the chromosome after n steps is obtained by integration over the appropriate interval of a normal density function that has a mean at the position of att and a standard deviation, a, of (n)'/2 (29). f we further assume that resolution occurs at the nth step, this is also the probability of a crossover in the interval. We have attempted to fit the data of Fig. 2 to this distribution, using different values of n, but have been unsuccessful: the frequency of region crossovers is approximately 15-fold greater than that predicted by the value of n that gives the best fit for the observed frequency of region crossovers. This disagreement is not resolved by assuming that resolution of a Holliday structure is random rather than fixed in time (C. M. Steinberg, personal communication). Two limiting hypotheses can account for this. (i) All site-specific recombination occurs with formation of crossed-strand exchanges within the core, but such exchanges have a disproportionately high probability of resolution (perhaps by int action) before they migrate into region. (ii) intpromoted crossovers only rarely lead to formation of crossedstrand exchanges, but once formed their probability of resolution is uniform along the chromosome. f we restrict ourselves to those Holliday structures that have migrated into region, the standard deviation of the normal distribution that best fits the region points of Fig. 2 gives us an estimate of the average excursion. This is equal to 6-8 nucleotide pairs. The total number of steps (forward and backward) in the average excursion is this value squared or X 15 nucleotide pairs. Thompson et al. (1), measuring migration rates in vitro, found that this number of steps could occur in 1.5 min at 37C (4.2 min at 25'C). Where are the strand exchanges resolved after migration? The most straightforward hypothesis is that they are resolved outside of att. Alternatively, resolution may occur only at att; in this event, exchanges that had migrated outside of att would have to back-migrate for resolution to occur, and we must invoke repair of transient mismatches to account for the region recombinants. Moreover, our estimate of the average excursion would have to be increased. Further work is needed to decide between these alternatives. A Holliday structure can be resolved by cutting of either the outside or the inside pair of strands (Fig. 1). Our observation of region recombinants with both possible configurations of outside markers indicates that both modes of resolution in fact We note that the data of Fig. 2 were obtained with attp X attl crosses. Any Holliday structures formed within the core region in such crosses should be unable to migrate leftward because of nonhomology. For this analysis we assumed that the barrier imposed by the nonhomology is a reflecting barrier and modified the above function accordingly (29). Proc. Natl. Acad. Sci. USA 76 (1979) 1367 occur. Furthermore, our observation that the two configurations are not found with equal frequency suggests either that the resolution mechanism prefers to cut the outside pair or that some subsequent event reduces the relative viability of the products that result from cutting the inside pair. Such an event might be differential packaging of the two types of recombinant DNA molecules into phage particles. f, as we suggest, a strand exchange is produced during intpromoted recombination at a specific site and then can migrate prior to resolution, the dichotomy between generalized and site-specific recombination becomes less distinct. Others have suggested that specific sequences may act as signals for initiation or resolution of generalized recombination in prokaryotes as well as in eukaryotes (12, 3, 31). The degree of site specificity for a particular recombination system then becomes a matter of relative rates of initiation, migration, and resolution of strand exchanges. We are grateful to Frank Stahl and Max Gottesman for helpful suggestions and to Charley Steinberg for help with the statistical analysis. Mrs. Cora Harvey typed the manuscript. 1. Nash, H. (1977) Curr. Top. Microbiol. mmunol. 78, Weisberg, R., Gottesman, S. & Gottesman, M. E. (1977) in Comprehensive Virology, eds. Frankel-Conrat, H. & Wagner, R. (Plenum, New York), Vol. 3, pp Schwesinger, M. (1977) Bacteriol. Rev. 41, Gottesman, M. E. & Weisberg, R. (1971) in The Bacteriophage Lambda, ed. Hershey, A. D. (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY), pp Kaiser, A. D. & Wu, R. (1968) Cold Spring Harbor Symp. Quant. Biol. 33, Signer, E. 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