Proc. NatI. Acad. Sci. USAVol. 82, pp. 3776-3780, June 1985Genetics

Site-specific DNA inversion is enhanced by a DNA sequenceelement in cis

(site-specific recombination/recombinational enhancer/BAL-31 deletion analysis)

HANS E. HUBER*, SHIGERU IIDAt, WERNER ARBER, AND THOMAS A. BICKLEBiozentrum of the University of Basel, Department of Microbiology, Klingelbergstrasse 70, CH-4056 Basel, Switzerland

Contributed by Werner Arber, February 11, 1985

ABSTRACT A segment of the bacteriophage P1 genome,called the C segment, can be inverted by site-specific recombi-nation; the two different orientations of the invertible segmentconfer different host ranges to the phage. Inversion is cata-lyzed by the product of the cin gene which is adjacent to one ofthe crossover sites flanking the C segment. The Cin-catalyzedrecombination can be measured in trans by using tester plas-mids in which inversion switches on antibiotic-resistancegenes. We show here that an additional sequence, distinctfrom the two crossover sites, is needed in cis for efficient inver-sion. This sequence is part of the cin structural gene and stim-ulates recombinatlon more than 100-fold. We have localizedthe major enhancer sequence on a 72-base-pair fragment andfound its activity to be largely independent of the orientationor position of the sequence with respect to the crossover sites.

The site-specific DNA inversion system of bacteriophage P1is one of a family of prokaryotic DNA inversion systemswith recombinases that can complement each other (1-3).These include enzymes encoded by phage P1 gene cin (4, 5),phage Mu gene gin (1, 3), Salmonella typhimurium gene hin(6), and Escherichia coli gene pin (3). The Tn3/yS resolvaseis also homologous at the DNA level but does not comple-ment the DNA invertases (7, 8). Site-specific DNA inversionserves as a biological switch for gene expression. For phagesP1 and Mu, it controls the host range (9, 10), and for S. typhi-murium, it regulates the expression of flagellar antigens (2,6). In all the inversion systems studied, crossing-over occurswithin two inverted repeat sequences. The recombinasegene lies adjacent to one repeat and overlaps it with the cod-ing region for the carboxyl terminus (P1 cin) or with the pro-moter region (all other recombinases). In P1 the invertibleDNA segment, called the C segment, is 4200 base pairs (bp)long and is flanked by exceedingly long inverted repeats of600 bp. The two crossover sites, cixL and cixR, are muchshorter and are located at the outer ends of the repeats (4, 5).We have taken advantage of the fact that the DNA se-

quences between the two crossover sites of the C segmentare not essential for the recombination to construct testerplasmids for cin-mediated recombination (4, 11, 12). In theseplasmids the cin gene has been partially deleted and the Csegment has been replaced by a promoterless chlorampheni-col (Cm)-resistance gene which, after inversion, can be tran-scribed from a promoter outside the C segment (see Fig. LA).The system allows recombinase activity to be measured bothin vitro and in vivo with high sensitivity (11, 12). The inver-sion rates in vitro, however, were low compared to those ofsimilar systems constructed for the studies of gin and hinrecombinases (13-15). A comparison of the structure of thetester plasmids of these three systems showed that, due tothe different orientations of the recombinase genes, the re-gions encoding the amino termini of the hin and gin gene

products had been retained, whereas that of cin had beendeleted.We demonstrate here that the amino-terminal region of the

cin gene does contain a sequence that stimulates site-specificrecombination when it is present in cis. The activity of thisrecombinational enhancer sequence from phage P1, whichhas tentatively been named sis(Pl) (sequence for inversionstimulation), is largely independent of its orientation or of itsdistance from the crossover sites.

MATERIAL AND METHODSBacterial and Phage Strains. All cloning and DNA inver-

sion experiments were done in the E. coli K-12 strain WA921and its recA derivative WA3782 (16). Phages P1 and P1-15have been described previously (4).Recombinant Plasmids and Procedures. The construction

of the plasmid pHHL71 has been described (11) and its struc-ture is shown in Fig. lA. Plasmid pHHL127 (Fig. lA) wasobtained by cloning a Mlu I-Sph I fragment from the cingene between the Sal I and Sph I sites of pHHL71. Theblunt-end ligation of the filled-in Mlu I and Sal I sites re-created both sites. pHHL6 was constructed by inserting theBgl II fragment 5 from P1 DNA into the BamHI site ofpBR322 followed by a Sph I-Sph I deletion. The EcoRI-NruI fragment of pHHL71 in its Cm-sensitive configuration,which contains cixL, was replaced by the EcoRI-Bal I frag-ment of pHHL71 in its Cm-resistant orientation; the result-ing plasmid, pHHL131, has two cixR sites. Replacing theNru I-Mlu I fragment of pHHL127A86R (see Results) withthe Alu I-Mlu I fragment of pSHI210 (5), which carries theamino-terminal region of the cin gene but no promoter, gavepHHL136. pHHL132, a kanamycin-resistance plasmid withthe origin of replication of pl5A, overproduces the cin geneproduct from the lacUV5 promoter. It was obtained by clon-ing a Pst I-HindIII fragment from pHHL84 (11) between thePst I and HindIII sites of pGP222A (17). The ainpicillin-resistance gene then was inactivated by a short deletion atthe Pst I site.BAL-31 deletions were made after cleavage at unique re-

striction sites. After deletion, the DNA was circulatized byligation with a 10-bp EcoRI linker. The method of Maxamand Gilbert (18) was used for DNA sequencing.Standard Assay for DNA Inversion in Vivo. To assay for

DNA inversion, tester plasmids were used to transformWA3782 (pHHL132), which overexpresses the cin recom-binase from the lacUV5 promoter. After 3 hr at 37°C, ali-qUots of the cultures were plated onto LA plates (27) con-taining Cm (12 pg/ml) and ampicillin (200 ,g/ml) or ampicil-lin alone. The ratio of Cm-resistant to ampicillin-resistantcolonies was determined 24 hr later and served as a measureof inversion.

Abbreviations: bp, base pair(s); Cm, chloramphenicol.*Present address: Department of Biological Chemistry, HarvardMedical School, Boston, MA 02115.tTo whom reprint requests should be addressed.

3776

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Proc. NatL Acad Sci USA 82 (1985)

AE N SSp Ci L

pHHL71 1 II

) L_

B

E cixRB P E

cat /Z

E B pE

,300bpI

- IJ

(S M SIS (Pl) Sp

1/IA127 R

A120 RA 105 R

A90 RA86 R

A78 RA73R- A62R

pHHL127 - A18L- A19L- A26L

A28LA32LA36L

A44LA52L -

C

c0

ZD -0C o.C r

._0._

:0=C

(a00

End of deletion (cin gene coordinates)

FIG. 1. Localization of the sequence stimulating cin-mediatedsite-specific recombination. (A) Structures of the tester plasmidspHHL71 and pHHL127. Relevant elements include the truncatedcin gene, the crossover sites cixL and cixR (vertical bars), and thepromoterless Cm-resistance gene cat which, after inversion, is tran-scribed from the promoter (P) of the tetracycline-resistance gene.Sequences from pBR327 are indicated by thin lines. Restrictionsites: B, BamHI; E, EcoRI; M, Mlu I; N, Nru I; S, Sal I; Sp, Sph I.(B) The region with stimulatory activity, together with the BAL-31deletions. The crosshatched segment was later used for subcloning.The extent of the deletions is shown by thin lines, and the numberingcorresponds to the last bp of the cin gene which is still present. Allplasmid derivatives with deletions from the left also have the dele-tion A86R. The exact end points of the deletions A19L, A26L, 428L,A32L, A73R, A78R, A86R, A90R, and A105R were determined byDNA sequencing; the others were estimated by polyacrylamide gelelectrophoresis. (C) Relative inversion frequencies of the cat genein the deletion derivatives shown in B. The frequencies are givenrelative to those of pHHL127 (for deletions from the right) or ofpHHL136 (for deletions from the left), with 100% equal to 35% and23% inversion in the standard assay, respectively.

Table 1. Inversion of tester plasmids after in vivo exposure tothe ci gene product

% inversion in host(mean ± SD, n = 3)

Plasmid WA921(P1-15) WA3782(pHHL132)

pHHL71 <0.05 0.2 ± 0.1pHHL127 3 ± 0.5 35 ± 5

The Cin recombinase was provided in trans at the wild-type levelby a P1-15 lysogen or at a level >100-fold higher by pHHL132 (11).P1-15 is a hybrid phage carrying the cin inversion system of phageP1(4). Inversion was measured in the standard assay.

RESULTSThe Amino-Terminal Region of the cin Gene Enhances Re-

combination in cis. Fig. 1A shows the structures of two testerplasmids used to determine the influence of flanking se-quences on cin-mediated recombination. Inversion frequen-cies were monitored with the cin gene product supplied froma P1-15 lysogen or in a strain carrying the plasmidpHHL132, which overproduces the Cin protein (Table 1).Plasmid pHHL127 turned out to be more than 100 timesmore active in inversion than pHHL71, an enhancement thathas to be due to the different length of sequences derivedfrom cin. In pHHL127, the cin structural gene only lacks thefirst 17 bp; in pHHL71 it is another 188 bp shorter.BAL-31 deletion analysis was used to determine which

part of the additional sequence in pHHL127 was responsiblefor the stimulation of recombination. Fig. 1B shows an en-largement of the 188 bp of extra DNA in pHHL127 and indi-cates the deletions that were analyzed. Deletions from theright were made from the unique Sph I site in pHHL127. Thedeletion A86R then was used to construct deletions extend-ing in from the unique Sal I site on the left of the region. Theinversion frequencies of the different deletion mutants weremeasured in the standard assay and are shown in Fig. 1C.Deletions from the right passing position 86 of the cin geneand all but two short deletions of the very left end drasticallyreduced the recombination efficiency. To determine wheth-er sequences of the cin gene upstream of position 17 (Mlu Isite in Fig. 1A) would also contribute to the enhancing activi-ty, the cin gene in pHHL127A86R was extended for 35 bp.The resulting plasmid pHHL136 carries intact cin sequencesfrom position -18 to +86 (see Materials and Methods; num-bering with respect to the adenylate residue of the initiationcodon of cin). Inversion in pHHL136 is only about 10% morefrequent than in its parent plasmid pHHL127A86R. The dele-tions thus define relatively well the limits of the sequencethat enhances site-specific recombination in cis. More than80% of the maximum activity resides within a 72-bp se-quence starting at position 19 of the cin gene (Fig. 2).The Recombinational Enhancer Is Active in Either Orienta-

tion and at Different Distances from the Crossover Sites. Totest the dependence on position and orientation of the en-

10 Nlul V 30 40 50 60 70 V80 Alul 90 100

AAAAT ATG CTA ATA GGC TAT GTA CGC GTA TCA ACA AAT GAA CAA MC ACT GCT TTA CM CGA AAC GCT CTT GAA AGC GCA GGA TGT GAG CTA ATT TTT GAG GAC AAG GCG

CC:GA G:: ::G ::T ::: ::: ::: A:G ::: ::: ::: ::: ::C ::G ::T ::A :AC C:G ::: ::: ::: ::: ::: :TT T:T ::: ::: ::: ::A :A: ::A ::: ::A ::T ::A TTA

TC:TG GCT ACT ::T ::G::: A:T ::G ::G ::: ::: :T: ::C ::: ::T :TC :A: ::: ::G ::T ::T ::G::: ACT ::T::: AAT ::: ::C :GC::: ::: ::A::: CGT ATC

TT:: C ::: ::T ::T ::: ::: ::: ::: ::: ::: ::: ::: ::C ::G ::: ::A :A: C:: ::: ::T ::T ::G ::G AAC T: T ::: ::: ::C ::: ::G ::: ::: ::A ::: ::: ATA

lie Gly Tyr Arg Val Ser Thr Gin Asn Leu Gln Arg Asn Ala Leu

FIG. 2. Nucleotide sequence of the P1 enhancer compared with the corresponding regions of the Mu gin, S. typhimurium hin, and E. colipingenes. Nucleotides -5 to + 105 of the cin gene are shown in the upper line (5). The 72-bp sequence which carries the major enhancer activity isindicated by larger letters, and the 68-bp fragment used for subcloning is underlined. Triangles indicate the smallest deletions from the left andthe right, respectively, that abolish the stimulating activity completely. The sequences of the homologous recombinase genes are aligned to givemaximum match and start at positions -5 (gin, pin) and -2 (hin) (3, 19). Only bases in positions differing from the cin gene are shown; identicalbases are represented by colons. Amino acids conserved in all four genes are indicated below. Asterisks mark codons where the third position isconserved to the same extent as the corresponding amino acid; the probability of the conservation pattern is <2%.

Ala Cys

cin

ginhinpin

Ile Phe Glu Asp

Genetics: Huber et aL 3777

E S M Sp

pHHL127 sciE

-:- IM41

Proc. NatL Acad Sci USA 82 (1985)

E P Sp LNC EPvRB E

A PHHL71 CmrI I 1I

A

sis (P1)-1

sis (S.typh.)t750 bp

L R V sis(P1)-2E P Sp PvE BE

B pHHL 71 CmS I(eJI.fcV

R R V sis (P1)E H P N PvE B E

C pHHL131 I I I / /

V sis (P1)E Sp L M R B/Bg E

D pHHL 6 C-segmFIG. 3. Sites of insertion of the enhancer sequences in different

tester plasmids. The enhancer sequence from the P1 cin gene isshown by a filled triangle, and that from the S. typhimurium hin geneby an open triangle. The vectors were pHHL71 in its Cm-resistant(Cmr) orientation (A), pHHL71 (B) and pHHL131 (C) in their Cm-sensitive (CmS) orientations, and pHHL6 (D). pHHL6 is a pBR322derivative, the other plasmids derive from pBR327. Insertion deriva-tives were named pHHL71-sis(P1)-1 and pHHL71-sis(S. typhimuri-um) in A, pHHL71-sis(P1)-2 in B, pHHL131-sis(P1) in C, andpHHL6-sis(P1) in D. The approximate distances between the site ofenhancer insertion and the crossover sites cixR and cixL (marked Rand L) are 270 and 550 bp for pHHL71-sis(P1)-1, 170 and 650 bp forpHHL71-sis(S. typhimurium), 410 and 1220 bp in B and C, and 985and 3450 bp in D, respectively. Symbols are as in Fig. 1 except forthe truncated cin gene, which is crosshatched, and the following ad-ditional restriction sites: Bg, BgI II; H, HincII; Nc, Nco I; P, Pst I;and Pv, Pvu II. The C segment in D is drawn with a gap of 2000 bp.

hancer sequence, we subcloned a 68-bp fragment defined bythe deletions A19L and A86R, taking advantage of the EcoRIlinkers that were inserted during the deletion procedure.This fragment was inserted into EcoRI sites in three testerplasmids that lacked the enhancer sequence (Fig. 3). Single-copy insertions in both orientations were obtained for allthree plasmids. The distance of the inserts from the closestcrossover site ranged from 270 bp to 985 bp. The plasmidswere used to transform the Cin overproducer WA3782(pHHL132) and the extent of inversion was estimated by re-striction analysis after overnight growth (about 20 genera-tions). In all of the plasmids, the insertion of the enhancersequence stimulated inversion from less than 5% in controlexperiments to close to equilibrium (Fig. 4). Neither the ori-

A B C

Table 2. Inversion frequencies of tester plasmids with insertionsof the enhancer sequence sis(P1)

Relative inversionOrientation of frequency, %

Plasmid sis insert (mean ± SD, n = 3)pHHL127A86R Right 100 ± 11pHHL71-sis(P1)-2-L Left 51 ± 8pHHL71-sis(P1)-2-R Right 64 ± 12pHHL131-sis(P1)-L Left 38 ± 7pHHL131-sis(P1)-R Right 47 ± 10pHHL131-sis(P1)-LL Two inserts, both left 67 ± 17pHHL131-sis(P1)-LR Two inserts, inverted 54 ± 6pHHL71 No sis 0.8 ± 0.3pHHL131 No sis 1.2 ± 0.5

All insertions except for that in the plasmid pHHL127A86R are inthe EcoRI site of the vector pBR327. Tandem inserts are indicated.Plasmid nomenclature is as in Fig. 3, with an additional suffix for theorientation of the insert: right (R) indicates that the orientation of sisis the same as in the P1-15 genome and left (L) indicates invertedorientation (Fig. 3). Inversion frequencies are relative to pHHL127A86R, with 100% equal to 25% inversion in the standard assay.

entation nor the position of the enhancer sequence is impor-tant for stimulating recombination.The stimulation was quantitated for the insertions in

pHHL71 and pHHL131, which still have intact Cm-resist-ance genes; these frequencies are shown in Table 2. In allcases, the enhancer sequence sis stimulates recombinationat least 30-fold. The orientation of the insert and the choiceof the tester plasmid do not significantly influence the inver-sion rates. The subcloning of the enhancer, however, fromits original location in pHHL127A86R to the opposite side ofthe invertible DNA segment, reduces its activity by half.Tandem inserts of the enhancer sequence increase recom-

bination efficiency (Table 2). We have, therefore, measuredthe effect of multiple insertions in the plasmids pHHL71 andpHHL131. Fig. 5 demonstrates the positive correlation be-tween the number of insertions of the enhancer sequenceand the inversion frequency. Because we have found thatderivatives with inverted repeats of the sequence have a low-er plasmid copy number, the values for plasmids with directrepeats and inverted repeats are shown separately. It isclear, however, that plasmid copy number does not affectthe phenomenon.The S. typhimurium hin Gene Contains a Sequence That En-

hances cin-Mediated Recombination. Since the site-specificrecombinases Cin and Hin show a great deal of sequence

D E+ +-_-_ + +-_-_ + +-_±_+---_ ++-_-_-

_ - s s

bc

ab

ba

c

baab

a

a

b

c

ba

ab

FIG. 4. Restriction analysis of tester plasmids after in vivo exposure to the Cin recombinase. The plasmids were used to transform E. coliWA3782(pHHL132). After overnight growth, plasmid DNA was extracted and analyzed by restriction endonuclease digestion followed byagarose gel electrophoresis. Bands were visualized after ethidium bromide staining. The plasmids are pHHL71-sis(P1)-2 (A), pHHL71-sis(P1)-1(B), pHHL131-sis(P1) (C), pHHL6-sis(P1) (D), and pHHL71-sis(S. typhimurium) (E) (see Fig. 3). Lanes + and -: tester plasmids with andwithout enhancer sequences, respectively. Arrowheads show the orientation of the enhancer fragment with respect to the orientation of theplasmids in Fig. 3. In lanes marked s, the corresponding vectors with the invertible segment in either orientation have been run as standards.Bands a and b designate restriction fragments originating from plasmids in the initial and inverted orientation, respectively. Bands c come fromthe Cin-producing plasmid, pHHL132. The restriction enzymes used were Pst I and Pvu II (A and B), HincII and Pvu 11 (C), EcoRI and Mlu I(D) and Nco I and Pst I (E). See Fig. 3 for restriction enzyme cleavage sites.

3778 Genetics: Huber et aL

Proc. NatL Acad Sci USA 82 (1985) 3779

50

C

.0 =30) d 1.1T

20-

10

1 2 3 4 5 6 7Number of sis insertions

FIG. 5. The effect of multiple insertions of the enhancer se-quence on inversion. The number of inserts into the rightmostEcoRI site of pHHL71 and pHHL131 (Fig. 3 B and C) and theirorientation were determined by restriction analysis. Insertions withonly direct repeats of the fragment are represented by open circles,and those containing at least one inverted repeat with filled circles.Inversion was measured as described in Materials and Methods.

homology and can complement each other (2, 5, 19), we rea-soned that a stimulatory sequence might also be presentwithin the hin gene. An Mbo II fragment spanning the pre-sumptive enhancer sequence sis(S. typhimurium) was re-moved from the plasmid pJZ121 (20) and inserted into thePvu II site of pHHL71 (Fig. 3A). This 146-bp fragment con-tains the sequence from position -38 to + 108 of the hingene. The fragment increased the inversion frequency to lev-els similar to that found with the P1 enhancer sequence, andthe stimulation was found with both orientations of the insert(Fig. 4E).

DISCUSSIONIn this paper, we describe the properties of a DNA sequencethat enhances site-specific DNA inversion in the bacteri-ophage P1 genome. The sequence, which is part of the cinrecombinase gene, is active only when it is present on thesame replicon as the invertible DNA segment. Its activitydoes not depend, however, on a particular orientation or lo-cation with respect to the recombination sites, and it is ac-tive in vivo as well as in vitro (11). Such enhancers seem tobe present in the recombinase genes of all the related DNAinversion systems. Requirements for sequences flanking thecrossover sites were first reported for gin-mediated inver-sion in phage Mu (21) and hin-mediated inversion in S. typhi-murium (15), and the presence of a position-independentstimulating sequence in the gin gene has been detected re-cently (R. Kahmann, personal communication).

Furthermore, the enhancer sequences appear to be mutu-ally exchangeable, like recombinases themselves. We haveshown here that the S. typhimurium hin gene contains in itsamino-terminal region a DNA sequence that enhances cin-mediated inversion in the P1 system. We have also observedthat cin-mediated inversion in the Mu system is stimulatedby the Mu-specific enhancer sequence (unpublished results).We suggest that these allelic enhancer sequences be called

sis (for sequence for inversion stimulation) with the origin ofthe sequence in parenthesis: e.g., sis(P1).

Position-independent enhancer sequences have not beenfound in other site-specific recombination systems such asthe resolution of Tn3/yS intermediates (8, 22), integration/excision of phage X (23), or cre-mediated resolution in P1(24). In contrast, chi sites stimulate general recombination atvariable distances (25). Transposition immunity (8), whichinhibits rather than stimulates transposition in cis, may rep-resent a related phenomenon.

The 72-bp enhancer sequence sis(P1) has been defined bydeletions from both ends. Some internal parts of the se-quence, therefore, may not be required for the activity. Thetwo ends of the sequence have no homology with each otherand the entire sequence contains no statistically significantinverted or direct repeats. A comparison of sis(Pl) with thecorresponding regions of the complementing recombinasesgenes hin, gin, and pin gives few hints as to which sections ofthe sequences are important precisely because the enhancersequences apparently code for an essential part of the pro-teins with 65-85% conserved amino acids (Fig. 2). However,conservation of the third position of codons in genes ex-pressed at a low level (26) may indicate functions of theDNA apart from coding. An appropriate comparison of thefour recombinase genes suggests that the functionally impor-tant sequences of the enhancer may be located at its ends(Fig. 2).The mechanism by which sis stimulates recombination is

unknown. It might function topologically, by direct interac-tion with the crossover sites, or it might serve as a bindingsite for proteins involved in recombination. Fold-back struc-tures are unlikely to be relevant, since tandem insertions ofsis in direct or inverse orientation are equally active (Table2).DNase "footprinting" (28) experiments with the active Cin

recombinase showed no protection of the sis sequence underconditions where the crossover site cixL is protected (datanot shown). However, the enhancer sequence may serve asthe binding site for a host-coded protein necessary for effi-cient recombination. A host-factor requirement has in factbeen found recently for DNA inversion catalyzed by thephage Mu Gin recombinase (R. Kahmann, personal commu-nication).

We are very grateful to Regine Kahmann, who discovered thephenomenon of enhancement in site-specific recombination in bac-teriophage Mu, for communicating results before publication. Wewould like to thank Christoph Nager, John Shepherd, and GordonLark for discussions and Theres Pripfl for technical assistance. Thiswork was supported by grants from the Swiss National ScienceFoundation.

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