Role for DNA homology in site-specific recombination

J. Mol. Biol. (1983) 170, 3 1 9 - 3 4 2

Role for DNA

Homology

in Site-specific R e c o m b i n a t i o n

The Isolation and Characterization of a Site Affinity Mutant of Coliphage ~. ROBERT A. WEISBERG

Section on Microbial Genetics, Laboratory of Molecular Genetics National Institute of Child Health and Human Development National Institutes of Health, Bethesda, MD 20205, U.S.A. LYNN W. ENQUIST t

Laboratory of Molecular Oncology, National Cancer Institute National Institutes of Health, Bethesda, MD 20205, U.S.A. CARL FOELLER~ AND ARTHUR LANDY

Division of Biomedical Science, Brown University Providence, R I 02912, U.S.A. (Received 1 March 1983, and in revised form 19 May 1983) Site-affinity (or saf) mutations change the specificity of prophage insertion. We have isolated a ,saf mutation of the bacteriophage ~ attachment site by inserting the phage chromosome into and then excising it from a secondary host attachment site. This causes reciprocal exchange of two seven base-pair segments (the overlap regions) that lie within the cores of the two sites. Since the two overlap regions differ from each other in nucleotide sequence, the recombinant sites are mutants. We have determined the effect of overlap region homology on recombination. We found that homology promotes integrative and excisive recombination. This suggests that the two overlap regions interact directly during recombination. The pattern of segregation of the saf mutation during site-specific recombination shows that it lies to the right of the point of genetic exchange about 95O/o of the time. This is a surprising result because ~ integrative recombination normally occurs by two staggered, reciprocal single-strand exchanges, one at each edge of the overlap region (Mizuuchi et al., 1981). Since saf lies within the overlap region, we might have expected that the point of genetic exchange would occur to the left of saf as often as to the right. We offer two models to account for this. (1) The mutation alters the location of one of the single-strand exchange points. (2) Efficient: and strand-specific processing of mismatched base-pairs changes the expected segregation pattern. Present address: Molecular Genetics, Inc., 10320 Bren Road East, Minnetonka, MN 55343, U.S.A. Present address: Biogen, 241 Binney Street, Cambridge, MA 02142, U.S.A. 319 0022 2836/83/300319-24 $03.00]0 © 1983 Academic press Inc. (London) Ltd. l[

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R. A. WEISBERG E T AL.

1. I n t r o d u c t i o n R e c o m b i n a t i o n between the attP site of bacteriophage ~ and the attB site of Escherichia coli inserts the D N A of the phage into the c h r o m o s o m e of its host (Campbell, 1962; for recent reviews, see Nash, 1981; Weisberg & L a n d y , 1983). Insertion requires the phage int and the host I H F proteins. Excision of the prophage requires the phage x i s protein as well. g p i n t and I H F p r o m o t e reciprocal single-strand exchange a t two specific positions within the homologous core regions of a t t P and attB (Fig. l; Mizuuchi et at., 1981). The points of singlestrand exchange are offset by a 7 bp~" " o v e r l a p " region. -7

~

o

__[C---~G ¢ T T T T T T A IGTIc G A A A A A A T __lctlgc

__~g

t t t ttta cgaaaaaat

c t t t TTTA cgaaaaaat

-~

+7 TACTA A TG IT ATGATTCAA

t

a

c

t

a

sic

t

a t g a t t ag l a

~t-attP t

g

1, T ACT atgAT

A TA G T T Gt_~ CAA, C

C T T T t t t a t a c t a sic t t g-GAAAAAAT

attB

C

ATGat

tgaac]

art L

attR

Fro. 1. Reciprocal strand exchange between attP and attB at the positions indicated by the small arrows inserts the phage chromosome into the bacterial chromosome to give a prophage with attL and attR at its left and right ends, respectively (see Mizuuchi et al.. 1981; and the Discussion). The top strand of each sequence has its 5' end at the left. Phage sequences are in capital letters, bacterial in lower case. The overlap region (see the text) is indicated by large bold characters. The bases adjacent to the core are boxed, and those adjacent to the a2tP core are shaded as well. The full extent of attB is indicated in the Figure, but attP extends from about -152 to +82 (Mizuuchi et al., 1981; Hsu et al., 1980), Sequence hyphens have been omitted for clarity. L a m b d a inserts its chromosome into attB a b o u t 1000 times more efficiently t h a n it does into the best secondary a t t a c h m e n t site on the E. coli c h r o m o s o m e (Shimada et al., 1972; this paper). Previous results point to the i m p o r t a n c e of specific D N A - p r o t e i n interactions in recombination between attP and attB. These include studies of gpint binding to attP and attB (Kotewicz et al., 1977; Kikuchi & Nash, 1978; Ross et al., 1979; Ross & L a n d y , 1983), and the observation t h a t ~80 and 21, lambdoid phages with insertion specificities different from t h a t of )~, fail to c o m p l e m e n t int and xis m u t a n t s of )~ ( G o t t e s m a n & Y a r m o l i n s k y , 1968; Zissler & Campbell, 1969; Kaiser & Masuda, 1970; Enquist & Weisberg, 1977a). In this p a p e r we show t h a t D N A - p r o t e i n interactions are not the sole d e t e r m i n a n t of site-affinity. First, we present evidence suggesting t h a t crossing Abbreviations used: bp, base-pair; kb, 103 bases or base-pairs.

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over between attP a n d a s e c o n d a r y a t t a c h m e n t site t h a t differs from attP a n d attB within the o v e r l a p region can occur a t either o f the two points o f single-strand exchange. W e also find t h a t these crossover points are p r o b a b l y t h e s a m e for excision as for insertion. These findings offer a m e a n s of mobilizing o v e r l a p regions by site-specific r e c o m b i n a t i o n , a n d have e n a b l e d us to cross a p a r t i c u l a r o v e r l a p region from a s e c o n d a r y site into attP and attB. T h e p h e n o t y p e of these r e c o m b i n a n t s suggests t h a t the o v e r l a p region p a r t i c i p a t e s in a h o m o l o g y d e p e n d e n t D N A - D N A i n t e r a c t i o n t h a t is an i m p o r t a n t d e t e r m i n a n t of siteaffinity. A p r e l i m i n a r y r e p o r t of this w o r k has been published (Mizuuchi et al., 1981).

2. Methods and Materials (a) Phage and bacterial growth The conditions and culture media for growth of phage and bacteria are given by Enquist & Weisberg (1977a) and Schrenk & Weisberg (1975). (b) Strains Bacterial and phage strains are described in Table 1. The construction of the bio936 substitution will be described in a subsequent publication (R. A. Weisberg, unpublished work). Lambda bio936 carries the bioA and bioB genes, the attR site and a complete phage int gene. (c) Construction of ~ insertions in g a i t Cultures of attB-deleted, galactose-sensitive strains (see below) were infected at multiplicities of about l0 phage/cell. After 15 to 20 min for phage adsorption, at least l08 infected cells were diluted in growth medium (Tryptone or L broth) and incubated with aeration for 4 to 6 h at 32°C in order to allow expression of the galactose resistance phenotype. The number of viable cells was then determined by spreading a sample on LB plates at 32°C, and the number of galactose-resistant, )~-immune cells by spreading a sample on MacConkey-galactose plates at 32°C together with about l09 W248 particles (Shimada et al., 1973). Infection with strain W248 kills cells not lysogenic for ~. More than 80o/o of the galactose-resistant, W248-resistant cells carried a ~ prophage located in the gal operon as judged by one of the following criteria: (l) gal + revertants regained )~ sensitivity; (2) P1 transductants to gal + regained )~ sensitivity (Shimada et al., 1973); or (3) some cured cells became galT+ (see section (d), below). The galactose-sensitive, attB-deleted strains we used to select gal insertions were RW599, which is galactose-sensitive because of a galE mutation, or RWI204, which is galactose-sensitive because of the galT-safP mutation. (d) Curing of ~ inserted in gaiT Exponentially growing cultures carrying ~cIts857 inserted in galT were shifted from 32°C to 40°C by dilution in order to induce int and xis expression. After 5 min at 40°C the cultures were chilled briefly in ice, returned to 32°C and incubated for 4 to 6 h. Samples were spread on LB plates incubated at 32°C to determine total viable cells, and on LB plates incubated at 42°C to determine the number of cured cells. (Cells lysogenic for ~cIts857 are temperature-sensitive because heating induces the expression of phage genes.) At least 20 colonies of cured cells were replicated with toothpicks to MacConkey-galactose or EMB-galactose plates and scored as gal- (red, good growth) or gal- (white, poor

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R. A. W E I S B E R G

ET AL.

TABLE 1 Strains Strain

Relevant genotype

A. E. colt strain,~ KS302 N99 N5574 N5813 R879 RW495 RW599 RW614 RW721 RW791 RW842 RW I 194 RW1200 RW 1 2 0 4 RW1246 RW1258 RW1401 RW 1463 RWI475 RWI476 RW 1518 RWI5t9 RW 1520 RWI521 RW16{~) RW1635 SA495 SA731 W3101 YM(! B. Phage 2 strai'~'~ B12 B252 B265 B266 B267 B269 B270 B271 B272 B274 G274 W2 W248 W249 Yl Y224 Y958 Y961 Y962 Y966

V(gal-bio) sup- galK2 ga1490 V8 attB + (2bio233 cIam 14 VHI) ga1490 V8 attB + (2bio233 clts857) bioA24 supE galE- V(pgl-bio) RW599 ()wits857) ira gaiT R879 (2bio V[int-PI Sam7 b21 RW599 (.~clts857) in g a i t (2 cryptic-842) in gaiT RW791 galE + galT-safP ffaIT-safP RW1194 bio + V(gal-uvrB) attP + RW495 V(galT-bioB) N5574 art B-saf(; N5574 gal + RW1463 gal + KS3t)2 (),hSO ins80 imm21 ) R879 tonA (2imm21) KS392 (2hSO i~s80 imm2) R879 lonA (2imm2) N5813 attB-saf(l ILW614 galE + bio + V(chID-pgl) 1= V8] V(gaI-J) attR + galT I .s~lpF lain2 h8O in*S0 at/P + altP-safG gal8 bio936 V(gagF-bioB) cl-ts gal8 bio936 attB + cl-ts gal8 bio936 attB-safG cI-ts bio936 attR + cl-ts bio936 attR-safG cI-ts hS0 V(att-inl)clgal8 blo936 V(galT-bioB) lmm434 hS0 V(att-red) cIhS0 ir~s80 gal8 int-am29 gal49 bio936 galK Sam7 galE Sam7 gaIK galT-safP galE Sam7

Source and/or reference

Shimada et al. (1973) M.E. Gottesmam W3102 M. E. Gottesman M. E, Gottesman DelCampillo-(!ampbell et al. (1967) HtYH × C600: thr + leu + tonA + Shimada et al. (19731 Shimada et al. (19731 R. Weisberg, unpublished This work Enquist & Weisberg (1976) ('21 -resistant RW791 Cured RW1635; this work Cured RW1194: this work PI transduetion of RW1194 This work D. Chattoraj; DJ48 From SA269 of Feiss et al. (19721 This work Selection for gal + at 32°( . Selection for gal + at 32°(! KS302 + B252 R879 lonA + 1312 KS302 + W249 R879 tonA + W2 This work PI transduetion of RW614 Feiss et al. (1972) Shapiro & Adhya (1969) Shapiro & Adhya {1969) Goldberg & Howe (19691 NIH Collection NIH Collection From Y994 From Y995 From YI013; see Methods and Materials From B267; see Methods and Materials From B267 Y1 × B269 YIO21 × B270 From tdel 1 of Franklin (1967) From Y1013 F. ,Jacob; 2 P a P a From tdel 9 of Franklin (1967) NIH collection F. Jacob; 2857 NIH collection This work Induction of RW614 Induction of RW614 This work

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T.~m.E 1 (co~ti~,led) Strain

Relevant genotype Y994 Y995 Y 1013 Y 1021 Y1024 Y 1025

Sam7

Source and/or reference

attP-.safG ~a-m7 gal8 bio936 attP-.safG gal8 attL ÷ 9aI8 attL-safG

N. Sternberg; Gotdberg & Howe (1969) This work D. Chattoraj, DKC144 S + revertant of Y995 Yl x B269 Y 1021 x B270

attB-safG

Shedtovsky & Brenner (1963) Messing (1983) This work

('. Other ph<~ges C21 Ml3mp7 M 13mpT-28J

t Phage 2 strains whose number is preceded by B carry imm21, those preceded by W or Y carry imm2 and those preceded by Y carry the tits857 temperature-sensitive repressor allele. growth) after overnight incubation at 32°C. To score the galT phenotype of cured )~ insertions t h a t had a galE mutation, the EMB-galactose plates were spread before replication with about l09 2ga149 (strain Y958) particles. This phage carries the galE gene and a part ofgalT (S. Adhya & R. Weisberg, unpublished experiments). I t recombines with galT-safP to give galT +, but the frequency is so low t h a t galE- gaIT-safP cured cells score as gal- while galE- galT+ cured cells score as gal + by this test.

(e) Prophage-host junction sequences in gaiT The prophage-host junction sequences of insertion 614 were determined essentially as described by Bidwell & L a n d y (1979) for insertion 791. Two lines of transducing phage, one carrying the left prophage junction (Y961) and the other the right prophage junction (Y962) were isolated from a heat-induced lysate of strain RW614. The first phage line was selected o11 the basis of its ability to transduce a 9aIK m u t a n t (galK2 in strain N99), and the second by its int- xis- phenotype (see Enquist & Weisberg, 1976) and by its ability to transduce a galT m u t a n t (galT1 in strain W3101). galK2 and galT1 lie to the left and right, respectively, of the secondary site in galT (Shimada et al., 1973). The Sam7 mutation was crossed into these lines by growth on a host carrying a cryptic Sam7 prophage (strain RW721) followed by screening for phage t h a t formed turbid plaques on a mixed bacterial lawn consisting of strains YMC (supF) and N99 (suppressor negative). (Inactivation of gene S simplifies the preparation of high titer phage stocks.) Multiple lysogens of these 2 phage lines were constructed in strain RW495 (see Enquist & Weisberg (1977a) for the screening procedure for multiple lysogens), and phage stocks prepared by heat induction as described by Sehrenk & Weisberg (1975). Restriction fragments containing the prophage-host junctions were isolated according to the previously described strategy and sequenced by the methods of Maxam & Gilbert (1977). (f) Sequence of galT-safP A transducing phage carrying the gal operon substituted for the att-int-xis region of )~ (Y909 of Marini et al., 1977) was used to lysogenize galT-safP derivatives of insertions 614 (RWI200) and 791 (RWI204). We expect insertion and subsequent excision of this phage chromosome to occur by homologous recombination in the gal region. In agreement with this expectation, when a gal + lysogen of each strain was heat-induced, approx. 50% of the phage in each lysate were gaiT- and failed to give gal + recombinants with either of the 2 original 9alT-safP strains. They also failed to recombine with a cryptic )~ prophage inserted at the galT site (RW842), but did give gal + recombinants when crossed with strains

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carrying gal mutations lying to the left (N99) or to the right (W3101) of the secondary site. I t therefore appears t h a t they carry the galT-safP mutation. High titer lysates of these 2 phage lines (Y966 and Y965, respectively) were prepared as described in section (e), above, and the region containing the mutation sequenced as described for galT+ (Bidwell & Landy, 1979). (g) Sequence of attP-safG Five independent lysates were prepared by heat induction of strain RW791, and one plaque from each lysate was amplified to give phage strains Y995D, E, F, H and I. The 500 bp HindIII-BamHI fragment t h a t contains attP was cloned from each strain into pBR322 to generate plasmids pCF1 through pCF5. Each plasmid was digested with HinfI, and the 317 bp art-containing fragment was isolated and labeled at the 5' terminus as described in section (o), below. Following cleavage with MboII the art-containing fragment was gel-purified and sequenced (Maxam & Gilbert, 1977) for each of the 5 independent isolates. (h) Tra*~duetion of gal and bio Overnight cultures of strains carrying a galK or bioA mutation (N99 or R879, respectively) were infected with phage and plated in non-nutrient top agar on Penassaygal-TTC or Minimal-Casamino acid plates, respectively (Shimada et al., 1975). Red gal + papillae were scored after 40 h incubation at 32°C and bio + colonies after 24 h incubation at 32°C. To distinguish plaques formed by phage carrying the gal8 substitution from those formed by non-transducers, we used strain KS302 or derivatives as a lawn on TB-gal-TTC plates (Enquist & Weisberg, 1976). gal8 phages form red-centered plaques, and nontransducers, colorless plaques. To distinguish plaques formed by strains carrying the bio936 substitution from non-transducers, we again used KS302 or derivatives as a lawn, and replicated plaques with toothpicks to Minimal-Casamino acid plates seeded with about l0 s cells of strain R,879 or derivatives. After overnight incubation, bio936 plaques were surrounded by a halo of heavy bacterial growth, while non-transducers were not. (i) The use of strain N5813 to determine insertion .specificity This strain carries an insertion mutation in the gal promoter region, a wild-type attB site and, to the right of attB, an insertion of a ~ fragment containing genes N, rex and elts857. At 32°C this strain is phenotypically gal- because of the defective gal promoter. At 42°C it is phenotypically gal ÷ because inactivation of the temperature-sensitive ). repressor allows gpN-modified transcription to initiate at the ),PL promoter and to read through attB into the gal operon. However, a heteroimmune prophage inserted at attB blocks gal transcription from PL, and so the strain remains gal- at 42°C (M. E. Gottesman & J. Auerbach, unpublished results). To determine insertion specificity, N5813 was infected at a multiplicity of approx. l0 phage/cell with imm21 derivatives of the strain to be tested. The imm21 repressor does not prevent transcription from initiating at the ),PL site, and ~imm21 has no homology with the resident ~ fragment. Samples of the infected cells were spread immediately after virus adsorption on MacConkey-galactose plates with or without l0 ~ B274 particles. Infection with B274 kills cells not lysogenic for ~imm21. After overnight incubation at 32°C (or 37°C), the plates are transferred to a 42°C incubator to inactivate the ), repressor. The red color t h a t indicates gal operon expression developed after 4 to 6 h of incubation at 42°C. White colonies on B274-seeded plates indicate insertion at attB; red colonies, insertion elsewhere. The frequency of insertion at attB (or elsewhere) is the ratio of the number of white (or red) colonies on plates seeded with B274 to the total number of colonies on unseeded plates. In some experiments a relative of N5813 t h a t contained cIaml4 instead of cIts857 was used (N5574). Surprisingly, this strain forms white colonies on MacConkeygalactose plates at 32°C and pink colonies at temperatures greater than 37°C. Otherwise,

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scoring for insertion specificity was the same as for strain N5813. To screen individual plaques for insertion specificity, N5813 was used as a lawn on Tryptone broth plates, and cells from the centers of the plaques were streaked with a loop on MacConkey-galactose plates seeded with about l09 B274 particles. Insertions at attB were distinguished from insertions elsewhere as described above. (j) Construction of attB-safG Lysogens of strains N5813 and N5574 with ~attP-safG imm21 (B266) inserted at attB were selected as described in section (i), above. Cultures of the lysogens were infected with phage G274 at a multiplicity of about l0 phage/cell (to provide gpint and gpxis), and gal + survivors selected on MacConkey-galactose agar. These survivors, which had invariably lost immunity to infection with ,~imm21, were tested for their ability to support reinsertion at attB as follows. Mini cultures of independently isolated cured cells were cross-streaked against ~attP-saf + imm21 and ~attP-safG imm21 on MacConkey-galactose plates, attB-safG strains gave a red intersection with ,~saf + and a pink intersection with ~safG. attB-saf + strains gave the inverse reaction. The plates were incubated at 32°C overnight, then shifted to 42°C for 4 to 6 h, We found 2 attB safG strains of the 150 tested (RW1476 and RW1600). (k) Construction of ~attB-safG We first constructed a )~ transducing phage (Y1013) carrying a complete gal operon (galS), the bioA and bioB genes (bio936) and wild-type attB (which lies between gal and bio) (R. Weisberg, unpublished results). The region extending from within galT to within bioB (including attB) was deleted from this phage by crossing it with the E. coil deletion m u t a n t RW1401. To do this the phage was first integrated into RWl401 by selection for bio + transductants, and then excised by induction. Most of the phage in the lysate carried the deletion. An imm21 derivative of such a phage (B267) was used to lysogenize an attB-safG derivative of strain N5574 (RW1476). RW1476 was induced, and a bioA transducing phage (B270) was isolated from the lysate. The art region of B270 was sequenced as described below. (I) Sequence of attB-safG B270 DNA was digested with restriction endonuclease SauIIIa, and the resulting fragments were cloned into the BamHI site of Ml3mp7 replicative form DNA as described by Messing (1983). B270 DNA was prepared from a concentrated and purified phage lysate as described by Silhavy et al. (1983). Restriction enzymes, DNA ligase, and M13mp7 replicative form DNA were purchased from Bethesda Research Laboratories and used according to the instructions of the supplier. The clone of interest (M13mpT-28J) was identified by plaque hxvbridization (Benton & Davis, 1977; Messing, 1983) using 3 2 P-labeled p B A l plasmid (2 x 10~to 4 x l0 s cts/min per ~g) as a probe. This plasmid, kindly provided by K. Mizuuchi, is a derivative of pBR322 containing an insertion of bacterial DNA extending from position - 8 4 to position + 8 5 around attB (K. Mizuuchi, personal communication). I t was labeled by nick-translation essentially as described by Maniatis et al. (1975). The attB region of M13mpT-28J was sequenced by the method of Sanger et al. (1977) as described by Messing (1983). Sequencing enzymes and reagents were purchased from Bethesda Research Laboratories. The sequence of the top strand from - 1 l0 to + 3 4 was determined. (in) Measuring recombination in ~gal x ).bio and in )~× )~gal-bio crosses (i) The frequency of recombinant DNA molecules Strain KS302 was grown in L broth supplemented with 0"2% (w/v) maltose and l0 -2 MMgSO4. Exponentially growing cells were chilled in ice and infected with approx.

32ti

R . A . W E I S B E R G ET A L.

l0 phage/cell of each parent. After 15 min for adsorption, a portion of the intected culture was rapidly warmed to 36°C by mixing with prewarmed broth and incubated with shaking. At intervals samples of l0 s to 2 × 10 s cells were removed and rapidly chilled in ice. DNA was extracted and purified as described by Silhavy et al. (1983) and digested with endonuclease EcoRI to completion (as monitored by electrophoresis of EcoRI-treated infected cell DNA containing added 2 DNA). Approximately one-third of each sample was subjected to electrophoresis in a 0"8% (w/v) agarose gel in 40 mM-Tris, 20 raM-sodium acetate, 2 mMEI)TA (pH 7-9). The DNA fi'agments were transferred to nitrocellulose paper by blotting and hybridized to 32P-labeled pBB105 DNA (2 × 10 s to 4 x 108 cts/min per pg) as described by Silhavy et al. (1983). Plasmid pBB105, which was kindly provided by H. Nash, is a derivative of pBR322 containing a 1.6 kb EcoRI-BamHI fragment of attB. Hence, it will hybridize to fragments containing attB, attL or attR. The hybridized blots were autoradiographed, and the intensity of the bands was measured with a Quick-Scan Jr. densitometer. The band intensities on several radiograms exposed for different times were determined, and the ratios of recombinant to parental bands averaged. (iX) The frequency of recombinant phage particles (':ells infected as described above were diluted in L broth immediately after adsorption and incubated for 90 min at 36°C. After sterilization with chloroform, the lysate was plated on lawns of KS302 derivatives on Tryptone-galactose-TTC plates (see section (h), above). One KS302 derivative carried a prophage of the same immunity specificity as the 3ujal parent. Thus, only imm-gal + recombinants formed red plaques. Such recombinants can arise either by site-slmcific recombination at art or by general recombination in the region between bio and imm~/imm21. To determine whether the crossover had occurred at att or to the right of the bio substitution, the red plaques were tested for the presence of bio transducing particles as described in section (h), above. The fl'equency of the reciprocal recombinant was measured in the same way except t h a t a KS302 derivative carrying a prophage with the same immunity as the ,~bio parent was used. In this case the colorless (i.e. recombinant) plaques were tested for bio transducing particles in order to determine where recombination had occurred. (n) Cort,struction of ly,sogens and analysis by Southern hybridization L a m b d a cIts857 lysogens carrying the prophage at secondary sites were isolated by infection of strain KS302 with Y! as described by Shimada et al. (1972). Lysogens of 2attPsafG were isolated by infection of strain SA495 with Y995H in the same way. DNA was extracted and purified from 5 ml overnight cultures of these lysogens in L broth as described by Silhavy et al. (1983). The DNA was digested to completion with endonuclease EcoRI or H i n d I I l (see above), subjected to electrophoresis and transferred to nitrocellulose paper as described in section (m)(i), above. The immobilized DNA was hybridized with the Eco]~I C fragment, which was labeled with 32p by nick-translation (Maniatis et al., 1975). The C fragment contains attP and will therefore hybridize to prophage-host junction fragments. I t was obtained from EcoRI-digested 2gt, 2C (Thomas et al., 1974) and purified by agarose gel electrophoresis. The band formed by the 5.7 kb fragment was cut out of the gel and extracted by emulsification in I ml of 50 mM-Tris, 5 mM-EDTA (pH 7'5). DNA was purified from the emulsion by 2 successive phenol/chloroform ( l : l . v/v) extractions followed by precipitation with ethanol.

(o) Footprint The plasmid pCF2 (section (g), above) was used as a source of attP-safG DNA. The artcontaining HinfI fragment was isolated, 5'-labeled, digested with AvaI and repurified by gel electrophoresis to yield a singly labeled fragment extending from - 1 1 4 (HinfI) in the P arm to + 161 (Aval) in the P' arm. For the wild-type attP site a " p r i m a r y " H i n d I I + I I I restriction fragment was isolated from 2clts857 Sam7 DNA. From this, the att-containing

HOMOLOGY IN S I T E - S P E C I F I C R E C O M B I N A T I O N

327

HinfI fragment was isolated, 5'-labeled, digested with MboII and repurified by gel electrophoresis to yield the singly labeled fragment extending from - 1 1 4 (HinfI) in the P arm to + 173 (MboIt) in the P' arm. Labeling with [),-32p]ATP (New England Nuclear) and polynucleotide kinase (PL Biochemicals) was according to the procedure of Maxam & (filbert (1977). The conditions for incubation with purified int protein and partial digestion with neoearzinostatin have been described (Ross el al., 1979; Ross & Landy, 1982) and are l)ased on the "footprinting" technique of Galas & Schmitz (1978). The partial digests were electrophoresed in 40 cm 10% (w/v) acrylamide/8 M-urea gels at 1800 V for 4.5 h (attPsaf(') or 3'5 h (attP). (p) Plaque test for distinguishing ).attB-safG imm21 from 2attB + imm21 We plated the phage on a lawn of strain RW1258, a host that has attP substituted for attB. The centers of the plaques were replicated with toothpicks to an EMBO plate spread with 109 B274 particles in order to select for stable 2imm21 lysogens (Gottesman & Yarmolinsky, 1968). Lambda attB is insertion-positive and 2attB-safG is insertionnegative by this test. EW1258 was constructed as follows. SA731, a cryptic ~ lysogen deleted for the region, extending from gal to 2 gene J, was lysogenized with 2gal8 int am29 tits857 (Y224) by selection for 2-immune colonies as described by Shimada el at. (1972). A co-infecting heteroimmune phage was used to supply gpint. In order to delete the region from imm through attB, temperature-resistant survivors were selected, and these were screened for loss of the bio gene and for the ability to support integration of ,~altB + (which does not integrate efficiently into attB (data not shown}). RWl258 is one such strain. (q) Statistical analysis of insertion ,specificity We tested the hypothesis that the site-affinity of 2attP-safG is the same as that of wildtype 2 in a host lacking attB as follows. First we estimated the probabilit~ ~ of insertion at multiply occupied sites by their observed frequency in our total sample of 13 2attP-safG and 18 2 lysogens (Table 3). For example, the estimated probability of insertion at site E (Table3) is 4 / ( 1 3 + 1 8 ) = 0-129. The probability that no lysogens with site E occupied would be fimnd in our sample of 18 2 secondary site lysogens under the hypothesis that the site affinities of the 2 phages are similar is the zero-order term of the Poisson distribution: exp[-(0-129)(18)] = ()-098. The probability that neither site E nor site F would be occupied in a sample this size is the product of their separate probabilities, 9-6 × 10 -3. In a similar manne,', the probability that the 2attP-safG collection contains no lysogens with the B, D, (~ or cys sites occupied is ]-5 × l0 -3. These low probabilities lead us to reject the hypothesis (see Results).

3. Results (a) Isolation of s a f mutants A l t h o u g h 2 usually inserts its D N A at attB, it also inserts at s e c o n d a r y sites. T h e best o f these is a b o u t 0 . 1 % as efficient as attB ( S h i m a d a et al., 1972; see below). Sequence analysis of insertions into m a n y different s e c o n d a r y sites suggests t h a t crossing over occurs preferentially at either o f t w o positions within the attP core: - 3 / - 2 and + 4 / + 5 (Mizuuchi et al., 1981; W e i s b e r g & L a n d y , 1983). I f so, r e p e a t e d insertion o f 2 a t a given site m i g h t give t w o t y p e s o f p r o d u c t t h a t differ b y the location o f the crossover p o i n t (the p o i n t or region o f genetic exchange). We h a v e reported a galT insertion o f the + 4 / + 5 t y p e (insertion 791;

328

R. A. W E I S B E R G

E T AL.

Bidwell & Landy, 1979). The expected second type (insertion 614) was revealed by screening independently isolated insertions of I into the E. coli gal operon. Phenotypic tests that measured the correlation between prophage curing and reversion to galT+ (see section (e), below) showed that insertion 614 differed from 791. Genetic crosses (data not shown) suggested that both had occurred at the same or at very closely linked locations in gaiT. The nucleotide sequence of the prophage-host junctions of the two lysogens (Fig. 2) confirms these points. The crossover point in attP lies between positions - 3 and + 1 in insertions 614 and between + 4 and +5 in insertion 791. The location of the crossover point in the host chromosome is likewise shifted four to seven nucleotide pairs leftward in insertion 614 relative to insertion 791. The conservation of the alignment of the crossover points in attP and galT is striking and suggests that the two different insertions have occurred at the same secondary site. Since insertion into galT can occur by exchange at either of two pairs of closely spaced points, we suspected that excision can occur at either of these pairs of points too. If so, prophage excision from insertions 614 and 791 will generate mutations in galT and attP by reciprocally exchanging phage and host DNA in the region between the crossover points (the "overlap region"; see Fig. 2). galT

-7

~--qc

0

+7

t t t gTTTATACT

A A ~ - ~ m 4 - L

{C-~G C T T T t t t t caa

a a a[~-'~--

--I-¢--A-IGCTTTTTTATACTAA

I

6~4-R

- - attP

~

I

--[g--~c

t

t t g t t t t C a a a a a ~ - - g a I T

--[~-Ic

, t t g t t t t C a a T A A ~ ~ T m - L

[-~FC~G

C T m TTTTATAC

+ a a a{'~--~--~--79+-R

+ --[~--~c

t t t q T T T A T A. C. a . .

a a

~

-

-

gatT-sa~P

- - ~ o

c T m T t t t t C a a T A A

~

-

-

,,+P-safG

Fro. 2. Tile sequence of 2 2 insertions into gaiT and the generation of saf mutations. Crossing over between attP and galT (lines 3 and 4) at the points indicated by the left set of brackets produces the left and right prophage junctions of insertion 614 (lines 1 and 2, respectively), while crossing over at the right set of brackets produees those of insertion 791 (lines 5 and 6, respectively). Prophage excision from insertion 79t, if it occurs at the left-hand crossover point, produces a pair of saf m u t a n t s (lines 7 and 8; see the text). The nucleotide changes in the m u t a n t s are underlined. The type size and other conventions are those of Fig. 1 except that only the top strands are shown. The crossover point for insertion 614 is shown as occurring at position - 3 / - 2 . although the sequence is consistent with crossing over anywhere between - 3 and + I. The reason for this is explained in the Discussion. The sequences of 614-L and 614-R were determined as described in Methods and Materials, section (e). The sequences of 791-L, 791-R and gait are taken from Bidwell & Landy (t979).

HOMOLOGY IN SITE-SPECIFIC RECOMBINATION

329

mutants that arise by this mechanism should differ from wild-type by a 3 bp substitution. Indeed, prophage curing produced galT mutants that were detectable by plate tests (see below). The nucleotide sequences of two such mutants, one derived from insertion 614 and the other from insertion 791, had the expected 3 bp substitution in the overlap region (see Methods and Materials, section (f)). The reciprocal product of crossing over at these two points is an attP site with a host overlap region (Fig. 2). In fact, five independently isolated phage lines produced by induction of insertion 791 had the expected 3 bp substitution (Fig. 2; see Methods and Materials, section (g)). We conclude that the same two crossover points used for prophage insertion are also used for excision. We shall call the attP mutants safG (site-affinity, gal) and the reciprocal mutants in the galT site, safP (site-affinity, phage). The procedure we used to isolate saf mutants, a cycle of insertion and excision at a secondary site, was suggested by the work of Six (1963,1966) who isolated saf mutants of the unrelated temperate coliphage P2 in a similar way. Our first attempts to apply this method to ~ (Enquist & Weisberg, 1977b) were unsuccessful for several reasons. (l) Some secondary sites appear not to yield saf mutants; (2) the frequency of mutants for those that do is usually low among the excised phage (E. Appelbaum & R. Weisberg, unpublished experiments; see below); and (3) one of our original screening procedures discriminated poorly between saf and saf + (see below). (b) Insertion ,specificity of saf mutants Lambda attP-safG differs from witd4ype in its low frequency of insertion into attB. This was shown in two ways. First, 13 AattP-safG and five ~ wild-type lysogens were constructed by selection for immune colonies after infection of strain SA495. Lysates prepared from each of these 18 strains were screened for particles capable of transducing the gal and bio markers, which flank attB. All 13 lysates from ~attP-safG lysogens gave fewer than ten gal and bio transductants per ml of lysate, while all five lysates from wild-type ~ lysogens gave more than 2000 gal and 250 bio transductants per ml of lysate (see Methods and Materials). Second, ~ insertion into the attB site of strain N5813 blocks transcription of the gal operon, thereby rendering the host gal- (M. E. Gottesman & J. Auerbach, unpublished results; see Methods and Materials, section (i)). Most ~attP-safG insertions into this host remained gal ÷, while most A insertions became gal(Table 2). These results (and others not shown) show that ~tttP-safG inserts into attB about 0.05 to 0-5% as efficiently as does 4. The overall frequency of stable lysogens formed by ~attP-safG (both at attB and elsewhere) is about 2 to 20~o that of wild4ype )~ in an attB + host, but about 4 to 20 times that of wild-type ~ in an attB-deleted host (Table 2 and other data not shown; Shimada et al., 1972). Curiously, our standard plaque test for estimating efficiency of insertion (Gottesman & Yarmolinsky, 1968) indicated that AattP-safG was only marginally deficient in forming lysogens in a wild-type host, and the unreliability of scoring by this method accounts in part for our previous failure to detect safG mutants (Enquist & Weisberg, 1977b).

330

R. A. WEISBERG ET AL. TABLE 2

The effect of safG on a t t P function Frequency of insertion attB Other sites

,tattP-saf + AattP-safG

0.87 ~ 0.0(~6

< 0.002 0"018

Insertions at attB were distinguished from insertions elsewhere by using strain N5813 (Methods and Materials, section (i)). This test classifies as attB any multiple lysogens carrying prophages at other sites as well as at attB. The infecting phage strains were B265 (saf ÷) and B266 (.safG).

The prophage locations in ~attP-safG lysogens differ from those in typical secondary site lysogens of wild-type ~. The prophage-host junctions of the 13 ]ysogens of ,~attP-safG described above were examined by Southern hybridization (Southern, 1975). The sizes of the junction fragments produced by different restriction enzymes allow division of the 13 strains into two groups with four members each and five groups with one member each (Table 3). None of the seven groups resembled any of 18 independently isolated secondary site tysogens of wild-type )~. The 18 wild-type )~ lysogens can be subdivided into one group with three members, two groups with two members each and I I groups with one member each (Table 3). One of the latter groups (LE2008) is indistinguishable by Southern blot analysis from the "cys" group described by Shimada et al. (1972), which was represented by four members of a collection of 14 independently isolated lysogens (data not shown; see Table 3, footnote $). Statistical analysis of the distribution of insertion sites confirms the impression that the insertion specificity of )~attP-safG differs from that of wild-type ~ in an attB-deleted host (see Methods and Materials, section (q)). Lambda attP-safG also differs from wild-type )~ in its efficiency of reinsertion into galT (which is not a preferred secondary site (Shimada et al., 1973)): the mutant is five- to tenfold more efficient than wild-type (Table 4). The gatT-safP site can be distinguished from gaiT + by measuring its ability to support phage insertion, galT-safP is about 250-fold more efficient than galT + for insertion of wild-type )~, but only 25~/o as efficient for insertion of" ,~attP-safG (Table 4). These results suggest that both the match of the two recombining sites to the wild-type phage sequence and their match to each other increase the frequency of attPxgalT recombination. The effect of safG on attPxattB recombination (following section) is somewhat different and suggests that the match of the sites to the wild-type sequence is relatively unimportant in this case,

(c) The effect of safG on a t t P × attB recombination To cross safG from attP to attB, we inserted ,~attP-safG into attB and then e::cised it. The cured cells were screened for their ability to support reinsertion of a wild-type phage into attB (Methods and Materials, section (j)). A strain in which reinsertion of A into attB was inefficient was selected for further study. The

H O M O L O G Y IN S I T E - S P E C I F I C

RECOMBINATION

331

TABLE 3

C o m p a r i s o n o f A a t t P - s a f G lysogens to ,~ secondary site lysogens Size of junction fragment {kb)

EcoRI Group

I.eft

HindIII

Right

Left

Strain

A. Lambda attP-safG lysogens E E E F U1 U2 F U3 U4 E F F U5

5"1 5-2 5.2 9 9 5.2 9 [2 5 5.2 9 9 19

18 18 18 8 2-6 4-1 8 22 8 18 8 8 2-3

18 17 17 21 9-2 16 21 21 11 17 21 21 17

RW1343 RWl413 RWI414 RWI415 RW1416 RWl417 RWl418 RWl419 RWI420 RW1421 RW1422 RW1423 RW1424

30 17 26 3O 24 14.5 16 20 16 19 12 14-5 12.5 9-6 23.5 12.5 12-5 20

LE2000 LE2001 LE2002 LE2003 LE2004 LE2005 LE2006 LE2007 LE2008 LE2009 LE2010 LE201 l LE2012 LE2013 LE2014 LE2015 LE2016 LE2017

24

RW 1375

B. Lambda seco~utary site lysoger~ U6 U7 U8 U9 Ul0 I) UI 1 B Cys~ Ul2 Ul3 D G UI4 U15 G G B

8-1 7-5 4-9 14 8 20 1I t 4-8 16 17 22 20 17

12 2-4 10 5 7-2 3'5 5"7t 2.2 18 4.8 6'8 3.5 12 (18)§

25 17 17 4.8

4 12 12 2-2

C. Lambda primary site lyso!len A

5

8

Lysogens were classified into groups according to the sizes of their prophage-host junction fragments. These were determined by Southern (1975) analysis as described in Methods and Materials section (n). Groups designated U followed by a number have only one member. The left EcoRI junction fragment was distinguished from the right by the relative intensities of the bands: the left has al)out 3 times more homology to the probe than does the right and, in addition, cannot be smaller than 4 kb. The right junction fragment produced by digestion with HindIII has so little homology to the probe that it could not be reliably scored in these experiments. All of the 2attP-safG lysogens were in strain SA495 except RW1343, which was in strain N99. All of the 2 lysogens were in strain KS302 ex(,ept RW1375, which was in strain N99. t ]'his strain may be a multiple lysogen; hence, the 5"7 kb right junction fragment may be incorrectly identified. :~ This lysogen has junction fragments that are indistinguishable from those of lysogens 10, 62 and 66 in the cys group of Shimada et al. (1972) (see the text). A 4th lysogen in the Shimada collection was placed in the cys group because of its genetic properties. We estimate the frequency of the cys group for the purposes of the calculation described in section (q) of Methods and Materials as (1 +4)/(18+ t 4 + 13) = 0.111. § Only one EcoRI junction fragment was observed.

332

R. A. WEISBERG E T A L . TABLE 4 Insertion into the galT secondary site

Phage art P-saf + P-safG

Frequency of insertion in galT( × l06) Host art galT-safP gatT+ 100 ~l

~ 0.4 ~4

Strains carrying galT-safP (RW1204) or ga/T+ (RW599) were infected with phage carrying attP-saf ÷ (Y994) or attP-safG (Y995). Selection for gal insertions is described in Methods and Materials, section (c). The numbers in the Table are means of 4 to 6 experiments. There was considerable fluctuation in the 3 smaller numbers, but the relative order of the 4 frequencies was always as shown.

nucteotide sequence of the attB site of this strain (Methods and Materials, sections (k) and (I)) had the three-base substitution associated with attP-safG, and we therefore call it attB-safG. The frequency of insertion of ~ and ,~attP-safG into attB and attB-safG is shown in Table 5. Insertion was inefficient when the two overlap regions were mismatched at the bases affected by safG and efficient when they were matched. A more elegant version of this experiment, which yielded similar results, is shown in F i g u r e 3 , columns 1 and 2. In this case, both recombining sites were on phage chromosomes, and the frequency of recombinants was measured among the progeny phage particles after mixed infection. Appropriate controls (Fig. 3, columns 3 and 4) showed t h a t both chromosomes were capable of interacting in all four crosses. These results show t h a t match of the recombining sites to each other at the positions altered by safG promotes recombination of attP with attB. In contrast, the match of the sites to the wild-type sequence is relatively u n i m p o r t a n t . Recombination between attP-safG and attB-safG should yield attL-safG and attR-safG (see Fig. 3). The )~gal (attL) and ,~bio (attR) transducing phages t h a t issued from this cross were purified and crossed to each other and to wild-type attL and attR phages. We again found t h a t efficient recombination occurred only when the two recombining att sites had the same overlap region (Fig. 4). However, the decrease in recombination due to mismatch was less marked than in the attP × attB cross.

TABLE 5 Effect of safG on insertion into a t t B

Phage

Frequency of insertion in: att B-saf + att B-safG

~attP-saf + )~attP-safG

0-87 ~ 0-0006

~ 0.003 0-44

attB + (N5813) or attB-safG {RWI600) strains were infected with ~attP.saf + (B265) or ),attP-safG (B266), and insertions into attB were identified as described in Methods and Materials, section (i).

P

I --

~x/-

I I I I I

go/

1

B

blo

21 offB

offP

sol +

sofG

sof +

sofG

sol +

II / II

0.110.2

1.2/0.95

2-5/2.5

sofG

0.09/0.2

8.1 / 7.1

1,8/I-5

I-7/I.9

Integrative recombinoi'ion

General recornbinofion

FIG. 3. The effect of .safG on integrative recombination in a phage cross. Crossing over at the att sites is indicated by the pair of unbroken brackets, and in the homologous region between bio and imm21/imm2 by the pair of smaller broken brackets. The percentage of the former class of recombinant among total progeny is given in columns l and 2 (Integrative recombination), and of the latter class of recombinant in columns 3 and 4 (General recombination). The pairs of numbers separated by a slash are percentages of reciprocal recombinants: the numbers before the slash refer to imm2 and those after the slash to imm21 recombinants. The parental phage strains were Y1 (attPsaf+), Y1021 (attP-safG), B269 (attB-saf +) and B270 (attB-safG), and the procedure for crosses and scoring recombinants is described in Methods and Materials, section (m)(ii). gal

L

1

\fl

I R

21

b/O offR

ottL

sol +

safG

sof +

sofG

sol +

24151

5.918-1

cl / ~<2

0-96/1,7

aafG

2.915-4

16/55

0~35/(~99 c0.5/<~I

Excisive recombination

General recombinal'ion

FIG. 4. The effect ofsafG on excisive recombination in a phage cross. The details are described in the legend to Fig. 3 and in Methods and Materials, section (m)(ii). The parental phage strains were Y1024 (attL-saf+), Y1025 (attL-safG), B27I (attR-saf +) and B272 (attR-safG).

R. A. W E I S B E R G E T AL.

334

TABLE 6 The effect of safG on the production of recombinant D N A (;ross

Percent recombinant fragment

attL-,~af+ x attR-saf + attL-safG xattR-safG

0rain
10min 11 <6

20 rain 27 28

30min 36 32

This cross measures excisive recombination, and the parental phage strains are those used for the experiment of Fig. 4. Samples were removed at the indicated times, and the relative amount of a recombinant DNA fragment containing attB measured as described in Methods and Materials. section (m)(i). The radioactive probe was a plasmid containing an attB insert, and it therefore hybridized to fragments containing altB, attL and attR. The identity of the attB band was verified by e3ectrophoresis of an EcoR| digest of genuine attB recombinant DNA. The identities of the attR and attL fragments were deduced from the )~ restriction map (see Daniels et al., 1983) and the restriction map of ~gal8 (M. Irani & S. Adhya, unpublished experiments). We measured the kinetics of attLxattR (or excisive) r e c o m b i n a t i o n b y e x t r a c t i n g t h e D N A f r o m m i x e d l y i n f e c t e d cells a t v a r i o u s t i m e s a f t e r i n f e c t i o n a n d s u b j e c t i n g i t to S o u t h e r n a n a l y s i s to d e t e c t r e c o m b i n a n t D N A f r a g m e n t s . W e f o u n d t h a t attL x attR a n d attL-safG × attR-safG r e c o m b i n a t i o n o c c u r a t s i m i l a r r a t e s , a l t h o u g h t h e s e c o n d r e a c t i o n m i g h t be r e t a r d e d b y a few m i n u t e s ( T a b l e 6). T h e f r e q u e n c y o f r e c o m b i n a n t D N A w a s m u c h lower in crosses w h e r e o n l y one o f t h e p a r t n e r s c a r r i e d safG o r in crosses o f ~attP + w i t h ,~attB + ( d a t a n o t s h o w n ) , P r e v i o u s w o r k has s h o w n t h a t attP x attB (or i n t e g r a t i v e ) r e c o m b i n a t i o n is less efficient t h e n e x c i s i v e r e c o m b i n a t i o n ( c o m p a r e F i g s 3 a n d 4), o c c u r s m o r e s l o w l y ( N a s h , 1975; G o t t e s m a n & G o t t e s m a n , 1975), a n d r e q u i r e s a h i g h e r level o f gpint ( E n q u i s t et al., 1979). T h i s m a y e x p l a i n t h e low level o f r e c o m b i n a n t D N A a t e a r l y t i m e s a f t e r infection. W e c o n c l u d e t h a t t h e effect o f safG on e x c i s i v e r e c o m b i n a t i o n is s i m i l a r to its effect on i n t e g r a t i v e r e c o m b i n a t i o n . Since t h e t w o r e a c t i o n s a r e n o t s i m p l e c h e m i c a l r e v e r s a l s o f each o t h e r ( G u a r n e r o s & E c h o l s , 1970; E c h o l s & G u a r n e r o s , 1983), t h i s is n o t a t r i v i a l conclusion. (d) I n t protein binding to a t t P - s a f G B e c a u s e of t h e high f r e q u e n c y of s a f G × s a f G i n t e g r a t i v e a n d e x c i s i v e r e c o m b i n a t i o n , we e x p e c t t h a t gpint will b i n d n o r m a l l y to t h e m u t a n t sites. T h i s w a s c o n f i r m e d for attP-safG b y d e t e r m i n i n g t h e region p r o t e c t e d f r o m n u c l e a s e a t t a c k b y gpint b i n d i n g . W e were u n a b l e to d e t e c t a n y differences b e t w e e n t h e m u t a n t a n d w i l d - t y p e s i t e s in t h e region o f t h e core (Fig. 5). W e e m p h a s i z e t h a t Fro. 5. Int protein binding to attP+safG at the core and P' sites. Restriction fragments containing either attP-safG or attP + were each 5'-labeled in the top strand at the HinfI ( - 114) site of the P arm as described in Methods and Materials, section (o). Following incubation in the presence (+) or absence ( - ) of Int protein, each fragment was partially digested with the A+T-specific reagent neocarzinostatin (NCS) (see Ross et al., 1979; Ross & Landy, 1982) and electrophoresed on 40 cm acrylamide gels (Methods and Materials, section (o)). Similar concentrations of purified Int protein were required to visualize the nuclease-protected sites in the 2 restriction fragments, but small differences in relative affinities were not assessed.

H O M O L O G Y IN S I T E - S P E C I F I C

offP$of

RECOMBINATION

Wild - t y p e

Int +

Int" --

+

--

p, p~

Core

Core

FIG. 5.

335

R. A. WEISBERG ET AL.

336

this test is not q u a n t i t a t i v e and will not disclose small differences in binding constants. However, other work shows t h a t the nucleotides altered by safG are not p a r t of the consensus gpint binding sequences in the core region (Ross & Landy, 1983).

(e) Segregation of safG into recombinant progeny Mizuuchi et al. (198l) have shown t h a t recombination of attP with attB occurs b y two staggered, reciprocal single-strand exchanges (Fig. 1). I f recombination of a safP with a safG site proceeds in the same way, each r e c o m b i n a n t joint will have three m i s m a t c h e d base-pairs (Fig. 6). I f this i n t e r m e d i a t e is resolved by semi-conservative D N A replication, it will give rise to two t y p e s of r e c o m b i n a n t progeny in equal numbers: top, which has inherited the " t o p " strand sequence of the heteroduplex, and bottom, which has inherited the " b o t t o m " strand sequence (recombinants I and I I , respectively, of Fig. 6). R e c o m b i n a n t s I and I I , which are the p r o p h a g e - h o s t junctions of insertions 614 and 791, respectively, can be distinguished from each other b y measuring the correlation of p r o p h a g e curing with reversion to galT+: m o s t cured derivatives of insertion 614 b u t few of insertion 791 were galT+ (Table 7). (We discuss possible reasons for this difference below.) Using this test and others, we found t h a t recombination of safP with safG produces an excess of top recombinants, in contradiction to the model just presented. First, insertion of ), into galT g a v e 14 top r e c o m b i n a n t s of 15 tested, as -3~ 0 TTTTATACT -

+5

-

-

-

att P

A A A A T A T G A

_

g t t t t c a a a

_

caaaagtt

t

- -

ga/ T

inse~ion 1 g TTTATACT c a a a ag

T t t t t

Replication g T T T A T A C T C A A A T A T G A

g

t

t

t

t

c

c a a a a g t t A

a

t C a a a

AAAATATG

t A

a

T

t

l T t t t t caa a Aa aa ag t t t

--

Recombinant !

TTTTATAGa

--

Recombinant II

AAAATATG

t

Fro. 6. Insertion via a heteroduplex intermediate. The overlap region (large bold characters) plus one flanking base-pair on each side is shown for attP (upper case) and the gaiT site (lower case). Insertion is assumed to occur by staggered, reciprocal single-strand exchanges at the positions marked by the 2 pairs of arrows. The resulting 2 heteroduplex intermediates, which would be located at the left and right prophage-host junctions, are resolved in this model by semiconservative DNA replication to give 2 pairs of recombinants in equal numbers (recombinant I and recombinant II). The first, which has inherited the sequence of the top strand of the heteroduptex, is identical to insertion 614, and the second, which has inherited that of the bottom strand, is identical to insertion 791. The mismatched bases in the heteroduplex intermediate are marked by over- and underlinings. Sequence hyphens have been omitted for clarity.

HOMOLOGY IN SITE-SPECIFIC RECOMBINATION

337

TABLE 7

Generation of saf mutants by prophage curing Curing frequency gal+/total 791 614

6"0 x 10-4 l-I x l 0 - 3

11/187 85/89

% gal+ 5"9 96

Cured cells were selected after a heat pulse and tested for galactose utilization as described in Methods and Materials, section (d). Insertion 791 was strain RW1246, and insertion 614 was strain RW 1635.

judged by the high correlation of curing with reversion to galT +. Second, insertion of ,~attP-safG into galT-safP gave 12 top recombinants of 13 tested (data not shown). (Recombinant I I is top in this cross because the sequences of the parental sites differ from those shown in Figure 6.) Measurement of excisive recombination disclosed a similar excess of top. We have already noted t h a t prophage excision from insertions 614 and 791 generates unequal numbers of gaiT + and gaIT-safP cured cells (Table 7). In both cases the top r e c o m b i n a n t is preferred. Since this recombinant was galT + for insertion 614 and galT- for insertion 791, phenotypic selection is not likely to account for the unequal recovery. Preferential recovery of the top recombinant m a y be even more extreme when the reciprocal p r o d u c t of prophage excision is examined: 189 of 192 phage produced by induction of insertion 791 were attP-safG (i.e. top) as shown by their inability to block gal operon expression after lysogenization of strain N5813 (Methods and Materials, section (i)). Finally, crosses between )~attL and ).attFt in which either one or the other parent was safG d e m o n s t r a t e d a similar preferential recovery of the top recombinant (Fig. 7). Thus, even when differences between the cores of the recombining sites were confined to the overlap region, unequal recovery was observed. We offer two explanations of the preferential recovery- of the top recombinant. (1) Recombination of safG with safG + sites proceeds via a heteroduplex

gQI

$ofG

go/

.......

sof +

( I ) oftB-sofG

I.......

'(2 ) o f f B - s o l + sol +

bio

( I ) ottB-sof

+

(2) o t f B - s o f G sofG

Cross I

Cross 2

Recombinon? ( I )

0/26

0/29

Recombinont ( IT )

26/26

29/29

bio

FIG. 7. The inheritance of safG in excisive recombination. Lambda gal-bio recombinants isolated from the attL-safG×attR-saf÷ (cross l) and the attL-saf+x attR-safG (cross 2) crosses described in Fig. 4 were isolated and purified. They were tested for the attB-safG character as described in Methods and Materials. section (p).

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intermediate, as shown in Figure 6, but the mismatches in the recombination joint are processed in such a way that bottom strand information is usually lost. (2) The differences between safG and safG + depress exchange of the bottom strands at the normal exchange point (position + 4 / + 5 ) , and resolution of the Holliday structure (Holliday, 1964) formed by exchange of the top strands occurs without the formation of heteroduplex DNA (see the Discussion). The first model demands that six mismatches (3 on each side of the prophage) be processed in favor of the top strand per recombination event. It has been proposed that the mulL and routS genes of E. coli are important ill processing mismatches (Glickman & gadman, 1980), and that adenine methylation carl direct processing so that the undermethylated strand is preferentially corrected in favor of the other strand (Radman et al., 1980). However, inactivation of mulL or routs by insertion mutations (Siegel et al., 1982) did not markedly change the preference ill favor of the top recombinant (data not shown). In addition there is no reason to think that the bottom strand is undermethylated relative to the top. Therefore processing, if it occurs, must be by some other pathway. We postpone detailed consideration of model (2) to the Discussion. 4. Discussion

The generation of saf mutants of the phage art site by a cycle of insertion and excision at a secondary host att site was first demonstrated for P2, a temperate coliphage not thought to be related to 2 (Six, 1963,1966; Calendar et al., 1976). Six (1966) speculated that the mutation arose by a double crossover in a region of interrupted homology. The work here confirms this speculation for ~. We have also isolated and characterized the reciprocal mutant, the altered secondary site. Lambda attP-safG lysogenizes E. coli inefficiently and usually at sites different from those used by wild-type )[. By contrast, a host carrying attB-safG is lysogenized with near normal efficiency at the altered attB site by the mutant phage. Wild-type ;t lysogenizes the mutant host poorly. The simplest interpretation is that insertion requires a direct DNA-DNA interaction between the two att sites, and that this interaction is promoted by homology in the region altered by safG. Other data (Fig. 4 and Table 4) support the same interpretation for excisive recombination. An alternative hypothesis, that protein binding to the altered site induces a specific conformational change in the complex such that it recombines only with a similarly changed complex, is unlikely because a different saf mutant fails to recombine efficiently with safG (E. Appelbaum & R. Weisberg, unpublished experiments). As more examples become available, we expect that the alternative hypothesis will become untenable. What is the nature of the DNA-DNA interaction? Kikuchi & Nash (1979; see Nash et al., 1981) have suggested that recombining art sites synapse to form a four-stranded structure before strand breakage occurs. Synapsis is favored by homology since it requires the formation of hydrogen bonds between pairs of identical base-pairs. Therefore, this might be the homology-dependent interaction. Alternatively (or in addition) the interaction could be the pairing of the

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complementary single-strands of the overlap regions after strand breakage has occurred. If the second model is correct, overlap region heterology might not interfere with synapsis or the initiation of strand exchange. In this event, safG × safG + recombination could well proceed to the Holliday structure stage (i.e. 2 strands reciprocally exchanged; Holliday (1964)) before stopping. (The arguments favoring a Holliday structure intermediate in site-specific recombination are summarized by Weisberg & Landy (1983).) However, measurement of integrative recombination in vitro detected neither Holliday structures nor recombinant DNA in heterologous crosses (Nash & Pollack, 1983). Substantial and comparable levels of recombinant DNA were found in both homologous crosses: saf + x saf + and safG ×safG. Therefore safG heterology prevents the formation and/or the accumulation of Holliday structures. There can be little doubt that the preferred locations of the crossover points (i.e. the points or regions of genetic exchange) in recombination between attP and secondary sites are the same as the locations of the single-strand exchange points in recombination between attP and attB. From analysis of A insertion into and excision from secondary sites, we infer that crossing over occurs preferentially at position - 3 / - 2 and at position + 4 / + 5 within the attP core {this paper; Mizuuchi et al., 1981; see Weisberg & Landy, 1983). Mizuuchi et al. (1981) showed that the points of strand exchange in attP × attB recombination are between bases - 3 and - 1 in the top strand and + 3 and + 5 in the bottom strand. N. Craig & N. Nash (personal communication) have recently shown that gpint cleaves two 5' sugar-phosphate bonds in attP and attB, one at position - 3 / - 2 in the top and the other at + 4/-I-5 in the bottom strand. Staggered single-strand exchanges in saf* ×safG crosses will generate a pair of heteroduplex overlap 'regions (Fig. 6). In the absence of mismatch repair, each heteroduplex should segregate one saf + and one safG recombinant. However, this prediction is contradicted by the results presented in Table 7 and Figure 7. Therefore, we prefer an alternative model in which safG heterology, in addition to its possible effect on the initiation of strand exchange (see above), also preferentially inhibits exchange of the bottom strands at the usual location. Since one of the altered base-pairs in safG is adjacent to the normal bottom strand exchange point, this assumption seems less artificial than that of strand-specific processing of mismatches. Reciprocal exchange of the two top strands at position - 3 / - 2 forms a Holliday structure (Holliday, 1964) in which branch migration rightwards is impeded by heterology between the overlap regions. Resolution of this Holliday structure into recombinant molecules might occur by exchange of the bottom strands at or near the position of the top strand exchange, and could be promoted either by gpint (Hsu & Landy, unpublished results) or by an E. coli enzyme analogous to endonuclease VII {Mizuuchi et at., 1982). The rare crossovers that do take place at the normal bottom strand exchange point could occur either by a similar mechanism that initiates with the bottom rather than the top strands, or by a pair of staggered exchanges followed by segregation (Fig. 6). In a variant of the model, suggested to us by a referee, resolution of the Holliday structure occurs by DNA replication rather than by a second strand exchange (see Golin & Esposito, 1981).

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AL.

It is interesting to compare a second core mutation, art24 (Shulman & Gottesman, 1973), to safG. art24 is a deletion of a T. A base-pair from the string of six that extends from position - 5 to 0 (Ross et al., 1982). Lambda attP24 differs from ~attP-safG in that it still prefers to insert its DNA at attB even though the overall frequency of insertion is depressed about 200-fold. The frequency of insertion is not increased in a host carrying attB24. Indeed, the art24 deletion, in contrast to the safG substitution, depresses the function of attB much more than that of attP (M. E. Gottesman & J. Auerbach, unpublished experiments). Excisive recombination of art24 with art + occasionally produces heterozygous phage particles. This argues strongly that recombination can occur by staggered single-strand breaks that straddle the att24 site. However, the pattern of segregation of the att24 mutation into recombinant progeny suggests that the mutant site usually lies to the left of the crossover point (Shulman & Gottesman, 1973; Nash et al., 1981). safG, in contrast, segregates as if it usually lies to the right of the crossover point (Fig. 7). Perhaps att24 preferentially depresses normal exchange of the top strands ( - 3 / - 2 exchange point) while safG preferentially depresses normal exchange of the bottom strands ( + 4 / + 5 exchange point) in crosses with wild-type sites. The effect of saf on recombination between attP and the secondary site in ga/T differs from its effect on attP x attB recombination: the frequency of safG x safG relative to saf + × saf + is depressed more when gaiT is the partner. We have no entirely satisfactory explanation for this observation. It may be that safG does depress protein binding to art, and that the depression is more severe (or more apparent) when bases outside of the overlap region differ from the primary site sequence (as they do in galT). The frequency of attP-safG×attB-safG recombination is reproducibly about one-third to two-thirds that of the wild-type frequency, and this may reflect an effect of the mutation on a DNA-protein interaction. The preferred sites of insertion of ,~attP-safG differ from the preferred secondary sites of wild-type )~. Secondary sites appear to be attB variants (Shimada et al., 1972,1975; Weisberg & Landy, 1983). The functionally important elements of attB consist of the overlap region and two gpint binding sites spaced 7 bp apart on either side (this paper; Ross & Landy, 1983). The two most active secondary sites that have been sequenced, gaIT-safP and proA/B (Pinkham et al., 1980), have phage-type overlap regions, but at least one poor or improperly spaced binding site. It seems likely that the preferred sites for insertion of )~attP-safG will have the same requirements for gpint binding as does attB, but will resemble safG rather than safP in their overlap regions. Their gpint binding sites may conform more closely to the consensus binding sequence and/or the canonical spacing than do those of the best secondary sites: the frequency of insertion of ~attP-safG at its best site is more than five times that of ~ insertion at its best secondary site. These speculations await confirmation by the establishment of a rigorous relation between structure and function for attB and its variants. att site recombination of )~ and of P2 superficially resemble each other and several other "conservative" site-specific recombination systems (Campbell, 1981). These pathways appear to have mechanistic features in common but differ in substrate specificity (see Weisberg & Landy, 1983). saf mutations change

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substrate specificity, and such changes have probably been i m p o r t a n t in evolution. I t is worth noting t h a t this kind of change in specificity will enable a single enzyme to promote efficient recombination within different groups of mutually compatible sites without promoting efficient intergroup recombination. The differences between some conservative p a t h w a y s v e r y likely include e n z y m e substrate as well as s u b s t r a t e - s u b s t r a t e interactions (see Introduction), but we have y e t to isolate mutations t h a t change e n z y m e specificity. Campbell's (1962) model for lysogeny assumes a role for homology in prophage insertion, and this assumption was invoked by Six (1966) to explain the phenotype of P2 saf mutants. The Campbell model predates our appreciation of the distinction between site-specific and homologous recombination (Signer & Beckwith, 1966). Nevertheless it is possible t h a t homology acts in similar ways in the two pathways. Enlightenment may come from the answers to two i m p o r t a n t questions about mechanism in each pathway: does a homology-dependent interaction occur before strand breakage, and does it involve anything other than Watson-Crick base-pairing? We are grateful to Ron Hoess for collaborating at an early stage of this work, to Wilma Ross for carrying out the nuclease protection experiments, to Ed Appelbaum, Glen Evans, Mary Jane Madden, David Margulies and Jon Seidman for help with sequencing and blotting and to Nancy Craig, Max Gottesman, Howard Nash and Nat Sternberg for reading and criticizing the manuscript. We are especially indebted to Max Gottesman for providing us with newly constructed strains and unpublished information. The work at Brown University was supported in part by grants from the National Institutes of Health (PHS AI 13544) and from the National Foundation, March of Dimes (1-543). REFERENCES Benton, W. & Davis, R. (1977). Science, 195, 180-182. Bidwell, K. & Landy, A. (1979). Cell, 15, 397-406. Calendar, R., Six, E. & Kahn, F. (1976). In Plasmids, DNA Insertion Elements and Episomes (Shapiro, J., Eukhari, A. & Adhya, S., eds), pp. 395-402, Cold Spring Harbor Laboratory Press, Cold Spring Harbor. Campbell, A. (1962). Advan. Genet. 11, 45-101. Campbell, A. (1981). Cold Spring Harbor Symp. Quant. Biol. 45, 1-9. Daniels, D., Schroeder, J., Szybalski, W., Sanger, F., Coulson, A., Hong, A., Hill, D., Petersen, G. & Blattner, F. (1983). In Lambda I I (Hendrix, R., Roberts, J., Stahl, F. & Weisberg, R., eds), pp. 515-676, Cold Spring Harbor Laboratory Press, Cold Spring Harbor. DelCampillo-Campbell, A., Kayajanian, G., Campbell, A. & Adhya, S. (1967). J. BacterioZ. 94, 2065-2066. Echols, H. & Guarneros, G. (1983). In Lambda I I (Hendrix, R., Roberts, J., Stahl, F. & Weisberg, R., eds), pp. 75-92, Cold Spring Harbor Laboratory Press, Cold Spring Harbor. Enquist, L. & Weisberg, R. (1976). Virology, 72, 147-153. Enquist, L. & Weisberg, R. (1977a). J. Mol. Biol. 111, 97-120. Enquist, L. & Weisberg, R. (1977b). In DNA Insertion Elements Plasmids and Episomes (Bukhari, A., Shapiro, J. & Adhya, S., eds), pp. 343-348: Cold Spring Harbor Laboratory Press, Cold Spring Harbor. Enquist, L., Kikuchi, A. & Weisberg, R. (1979). Cold Spring Harbor Symp. Quant. Biol. 43, 1115-1120. Feiss, M., Adhya, S. & Court, D. (1972). Genetics, 71, 189-206. Franklin, N. (1967). Genetics, 57, 301-318.

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