Proc. Natl. Acad. Sci. USAVol. 92, pp. 1352-1356, February 1995Cell Biology

The d4f resolvase locus of the Escherichia coli chromosome canbe replaced by a 33-bp sequence, but function depends on location

(terminus/deletion/inversion)

MARIANNE TECKLENBURG, ANNE NAUMER, OLAGAPPAN NAGAPPAN, AND PETER KUEMPEL*Molecular, Cellular and Developmental Biology Department, University of Colorado, Box 347, Boulder, CO 80309

Communicated by William B. Wood, University of Colorado, Boulder, CO, October 14, 1994

ABSTRACT The dif locus (deletion-induced filamenta-tion) of Escherichia coli is a resolvase site, located in theterminus region ofthe chromosome, that reduces chromosomemultimers to monomers. In strains in which this site has beendeleted, a fraction of the cells is filamentous, has abnormalnucleoid structure, and exhibits elevated levels of the SOSrepair system. We have demonstrated that a 33-bp sequence,which is sufficient for RecA-independent recombination andwhich shows similarity to the cer site ofpColEl, suppresses theDif phenotype when inserted in the terminus region. Flankingsequences were not required, since suppression occurred instrains in which difas well as 12 kb or 173 kb ofDNA had beendeleted. However, location was important, and insertions at asite 118 kb away from the normal site did not suppress the Difphenotype. These sites were otherwise still functional, andthey exhibited wild-type levels of RecA-independent recombi-nation with dif-containing plasmids and recombined withother chromosomal dfsites to cause deletions and inversions.It is proposed that the functions expressed by a difsite dependon chromosome location and structure, and analysis of thesefunctions provides away to examine the structure ofthe terminusregion.

Termination of the replication cycle is a key event in thebacterial cell cycle (1). Termination produces two completedaughter chromosomes, which can then be segregated to thedaughter cells during the ensuing cell division. Separate nucle-oids that contain the newly formed chromosomes appear aftertermination (2), and an essential aspect of separation is theremoval of DNA structures that link these chromosomes (3).One cause of joining of daughter chromosomes is catenation,and several type I and II topoisomerases capable of unlinkingcatenated chromosomes have been identified in Escherichiacoli (4, 5).An additional cause of joining of daughter chromosomes is

recombination. If an odd number of recombination events hasoccurred between the growing daughter chromosomes (sisterchromatid exchange), the completed chromosomes will bejoined as a circular dimer (6, 7). This structure cannot bepartitioned to two daughter cells unless a compensating re-combination event occurs, and topoisomerases do not nor-mally carry out recombination. A RecA-independent resolvasesystem that effects this recombination has been identified in E.coli (6-8). The resolvase site is called dif (deletion-inducedfilamentation) and is located in the terminus region at 34.2min, or 1600 kb on the physical map (9). The xerC and xerDgenes encode the resolvase proteins which function at the difsite, as well as at the cer site of the ColEl plasmid (7, 10).The dif locus was identified by the abnormal phenotype

associated with deletions of this part of the terminus (6). TheDif phenotype included filamentation of a fraction of the cells,abnormal nucleoids, and induction of the SOS response. This

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement" inaccordance with 18 U.S.C. §1734 solely to indicate this fact.

phenotype could be suppressed by integration of plasmids thatcontained a 530-bp fragment from the diflocus. This fragmentcontained a 33-bp sequence thatwas similar to the cer resolvasesite of ColEl, and the 33-bp site promoted RecA-independentrecombination (6, 7). It was ambiguous, however, whetherintegration of plasmids that contained this 33-bp sequence wassufficient to suppress the Dif phenotype.The studies reported here demonstrate that integration of

the 33-bp sequence, in the absence of plasmid sequences orflanking DNA from the dif locus, suppressed the Dif pheno-type, but only when inserted at the normal location for dif.Suppression also occurred when difwas combined with certaindeletions, one of which removed 173 kb of flanking DNA.Comet et al. (11) have recently replaced difwith psi, which isthe cer-like site of plasmid pSC101, and demonstrated that itsuppressed the phenotype of a 58-bp deletion.

MATERIALS AND METHODSMedia and Solutions. LB and M9 media have been de-

scribed (12). All media were supplemented with thymine (50,Lg/ml). M9 glycerol/Casamino acids medium contained 0.2%glycerol, 0.5% Difco Casamino acids, and tryptophan (20,ug/ml). Antibiotics were used at the following concentrations:tetracycline (Tc), 20 ,ug/ml; chloramphenicol (Cm), 25 ,Lg/ml;kanamycin (Km), 100 ,ug/ml; ampicillin (Ap), 100 ,ug/ml;spectinomycin (Sp), 100 ,ug/ml.

Bacterial Strains. PK2649 was a A(argF-lac)205 AsfiA::lacZ (13) derivative of PK457 (6). Other strains werederived from PK2649 by Pi transduction; A2336 was intro-duced by transduction and selection for KMr, to give PK2650.

Construction of dif3044. In order to form the spc-difcassette, a 2-kb blunt-endedHindIll fragment which containedthe spectinomycin-resistance gene (spc) of pHP45-omega (14)was inserted into the Sma I site of pUC18. The 33-bp difsequence,- which was isolated as a HindIII-BamHI fragmentfrom pMIN33 (6, 7), was inserted into the corresponding sitesof the polylinker to give pPK3011, which contained EcoRI-spc(->)-dif(--)-BamHI-HindIII (the arrows indicate the di-rection of spc transcription and the dif sequence; see below).A Tn9O3 kan cassette in the EcoRI site of pBR322 was thenopened at the Sma I site, and the blunt-ended spc-dif(EcoRI-HindIII) fragment from pPK3011 was inserted to givepPK3015. The kan::spc-dif cassette was then inserted intopMAK705. This temperature-sensitive derivative of pSC101(15) was digested with EcoR47III to remove the 0.5-kb frag-ment that contained a cer-like site (6, 11), the cam gene wasremoved in a 1.9-kb Xmn I-Nru I fragment, and the amp-containing 1.0-kb BspHI fragment of pBR322 was inserted togive pPK708. Finally, the kan::spc-dif construct frompPK3015 was inserted as an EcoRI fragment, to give pPK730.Integration of spc-dif into the chromosome and loss of theplasmid were obtained by isolating Spr Aps cells after growth at42°C of PK2649 carrying pPK730.

*To whom reprint requests should be addressed.

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Construction of dif3185 and dif3186. pJH142 contains a7-kb Hindlll fragment (from kb 1479 to kb 1486) in pA-CYC177. The 2.0-kb EcoRI-HindIII spc-dif fragment wasisolated from pPK3011, the ends were filled in, and theblunt-ended fragment was inserted at the Pvu II site of theHindIII fragment in pJH142. Both orientations were obtained.The resulting plasmids (pPK813 and pPK814) were digestedwith EcoRI and spc-difwas inserted into the chromosome ofPK3144 by linear transformation (16). PK3144 is a xerC::cam(17) recD::TnlO derivative of PK2650, and PK3185 andPK3186 are transformants with different orientations of spc-dif, subsequently transduced to thyA+ recD+.

Construction of dif3547. pAN8 is a pUC19 derivative thatcontains a 6-kb HindIII fragment (kb 1523-1529) inserted atthe HindIII site, a 1.7-kb EcoRI-HindIII spc-diffragment withfilled-in ends inserted at the Xba I site, and an EcoRVfragment (kb 1702-1704) inserted at the Sma I site. The spcgene used in this construction was the 1.67-kbXmn 1-Hindlllfragment derived from pHP45-omega (14); otherwise, theconstruction of the spc-dif cassette was similar to that de-scribed above for pPK3011. The plasmid was transformed intothe poUts strain PK2430 (6), and integration into the chro-mosome and loss of the plasmid were obtained by isolating SprAps cells after growth at 42°C. For transduction into otherstrains, nth::kan (18) was introduced as a flanking marker bytransduction. The deletion was then transduced, by Kmrselection and scoring for Spr and manrA, into PK2993 to givePK3547. PK2993 is a PK2649 derivative with A2771 (Cmr).DNA Procedures. The 33-bp dif sequence is 5'-ATTGGT-

GCGCATAATGTATATTATGTTAAATCA-3', which is lo-cated at kb 1600 and oriented as *- on the physical map (ref.9; see Fig. 1). An end-labeled oligomer with this sequence wasused as the probe in the Southern hybridizations shown in Fig.2. Other DNA procedures have been described (19).Recombination with dif-Containing Plasmids. The proce-

dures for these tests have been described (6). All of the strainstested were recA56 derivatives, constructed by cotransductionof recA56 with srl-300::TnlO. The plasmids used werepMAK705 (15) and a derivative (pPK706) that contains the33-bp sequence inserted at the polylinker site (6).

"Double-dif" Strains. Construction of PK3302 and PK3343started with PK3185 (xerC::cam A2336 dif3185) and PK3186(xerC::cam A2336 dif3186). A2336 was removed by transduc-tion to Tcr (zde-234::TnlO) and cotransduction of Kms dif+.Transduction to xerC+ (select for ilv+, score for Cms) convert-ed them to unstable strains PK3302 and PK3343. PK3545 wasconstructed by starting with PK3186 and using transduction toincorporate dif3044 and then xerC+. Loss ofDNA between dif

sites caused loss of fdnG, which was scored on McConkeynitrate plates (20).

SOS-Induction Assays. Solutions and procedures for deter-mining sflA::lacZ expression were as described by Miller (12).Table 1 shows the average of at least three separate assays, andvalues were within ± 10%.

RESULTSConstruction of dif Insertions. The purpose of the experi-

ments described here was to determine whether the Difphenotype could be suppressed by integration of a short,dif-containing oligomer. We used the 33-bp sequence that wepreviously demonstrated was sufficient for plasmid-plasmidrecombination, as well as high-frequency, RecA-independentrecombination between plasmids and the chromosomal dif site(6). The sequence exhibits high homology between dif and cer,and it contains the binding site for the XerC/XerD resolvase(10). The deleted sites into which the 33-bp sequence wasinserted removed dif and 12 or 173 kb of flanking DNA.

dif3O44 occurred by a double crossover with a plasmid whichcontained the 33-bp sequence in a spc-dif cassette in themiddle of a kan gene. Integration occurred at the site of A2336,in which a kan gene had replaced 12 kb of terminus-regionDNA (from kb 1591 to 1603). dif3044 had the same orientationas the normal dif site (Fig. 1). We have previously demon-strated that dif functions in both orientations when present atthis site (ref. 6; O.N., unpublished experiments).We have also constructed a strain (PK3547; Fig. 1) in which

the 33-bp sequence replaced dif as well as 173 kb of theterminus region (from kb 1529 to 1702). A plasmid wasconstructed that contained the equivalent of this deletion, withspc-dif inserted at the site of the deletion. dif3547 was derivedfrom a double crossover with the chromosome.

In addition to insertions at difs normal site, we have alsoconstructed strains in which the 33-bp sequence was insertedin both orientations at kb 1482 (31.7 min; Fig. 1). Theseinsertions (dif3185 and dif3186) were obtained by linear trans-formation.

Verification of the Location of Insertion Sites. The geneticlocations of the insertions were verified by a variety oftransductions. For example, dif3044 was replaced at 100%frequency by integration of A2771 (see Fig. 1). Analogousresults were obtained when dif3044 was integrated into a straincontaining A2771. Strains containing dif-spc in the 173-kbdeletion (dif3547) were manAfdnG, and these properties weretransferred to recipients by transduction. Also, dif3185 anddif3186 showed 80% cotransduction with trg::TnlO.

min 32.1tr ::TnlO

kb 1480

trg

- dif3185.-d/13186

1520 1560i I

fdnG

34.2zde-264 ::Tn 10

1600 1640

dif TerC

36.3tus

1680 ,

nthTerB manA

o A2771o A 2336a3 dif3044

dff3547dff3303

dif35461 IA2038A2035

FIG. 1. Genetic and physical map of the terminus region (9) showing relevant loci, deletions, and dif alleles. Arrows at the dif sites show theorientation of the 33-bp sequence. Clockwise replication forks move from left to right in the figure.

-

1 4 i

1 4

I Ai 1 --7

I I

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The insertions were also tested by Southern hybridization.Lanes 1 and 2 of Fig. 2 show the dif-containingEcoRV fragmentsin dif+ and dif3044 strains. DNAs from double-dif strains thatcontain dif+ as well as dif3185 (lane 3) or dif3186 (lane 4) are alsoshown. The same double-dif DNAs digested with HindIII areshown in lanes 7 and 8. The fragment sizes correspond to thoseexpected, based on the physical map (9) and alterations associ-ated with the particular dif allele. dif3547 lacks homology toprobes from the deleted region, and spc-dif is in the expectedfusion fragment (data not shown).

Effects of Insertions on the Dif Phenotype. The various difalleles were transduced into PK2649 to determine their effecton filamentation, and Fig. 3 shows some representative phe-notypes. dif3044 (PK3044; Fig. 3C) suppressed the filamenta-tion due to A2336 (PK2650; Fig. 3B) to the level observed indif+ bacteria (PK2649; Fig. 3A). Suppression did not occur ina control strain in which only the spc gene had been insertedat the site of A2336 (data not shown). dif3547 also suppressedthe filamentation, and the phenotype was similar to that shownin Fig. 3A. dif3185 and dif3186 did not suppress filamentation,and their phenotypes were similar to that of PK2650 (Fig. 3B).The level of SOS induction due to the various dif alleles was

determined by using an SOS-inducible sfiA::lacZ reporter gene(13). As we reported previously, SOS is induced in difmutants,although this is not the primary cause of filamentation (6);ftsZ114 mutants, which are insensitive to the SfiA cell-divisioninhibitor, continued to form shorter filaments. Since filamen-tation was difficult to quantitate and the fraction of cells thatformed filaments as well as their lengths varied, SOS inductionprovided an alternative means to estimate suppression. Forcells growing exponentially in M9 glycerol medium, the levelof SOS induction in PK2650 (12-kb deletion A2336) was4.3-fold that expressed by PK2649 (dif+), and the level inPK3140 (155-kb deletion A2035) was 15.7-fold that of PK2649(Table 1). The 33-bp insertion in the 12-kb deletion (dif3044),as well as the insertion in a 173-kb deletion (dif3547) reducedthis to wild-type levels. dif3185 and dif3186 did not reduce SOSinduction.The levels of SOS expression were also determined in cells

growing more rapidly in M9 glycerol medium supplementedwith Casamino acids (Table 1). In this medium, A2336 ex-pressed SOS at 11-fold the wild-type level, and dif3044 reducedSOS expression to 2.1-fold that of wild-type. Suppression bydif3547was almost complete, whereas the insertions at kb 1482gave high levels of SOS induction (dif3185 and dif3186).

11.3-

8.6 -6.6 -

4.9 -

4.5 -

2.1 -

-8.6

-6.6

5.2

-3.8

-3.2

1 2 3 4 5 6 7 8 9 10

FIG. 2. Southern hybridization of DNAs from dif strains. DNAsdigested withEcoRV (lanes 1-6) or HindIII (lanes 7-10) were probedwith the end-labeled 33-bp dif oligomer. Lane 1, PK2649 (dif+); lane2, PK3044 (dif3044); lanes 3 and 7, PK3298 (xerC dif3185 dif+); lanes4 and 8, PK3299 (xerC dif3186 dif+); lanes 5 and 9, PK3302 (xerC+dif3185 dif+), in which inversion occurred; lanes 6 and 10, PK3303,derived from a deletion in PK3343 (xerC+ dif3186 dif+).

FIG. 3. Phase-contrast photomicrographs of various dif mutants.Cells were freshly grown to stationary phase in LB medium. (A)PK2649 (dif+). (B) PK2650 (A2336). (C) PK3044 (dif3044). (D)PK3546 (dif3546). (X425.)

Recombination at dif Sites. We tested the dif sites todetermine whether suppression was related to a high level ofRecA-independent recombination. These tests were done inrecA derivatives of the strains described above, which alsocontained pPK706 (6). This derivative of pSC101 is tempera-ture sensitive for replication, encodes a gene that confers chlor-amphenicol resistance, and also carries the 33-bp dif sequence.The rationale of the test was that, in order to confer chloram-phenicol resistance on cells incubated at 42°C, the plasmid mustrecombine with a dif site in the chromosome.The frequency of integration of the plasmid into the wild-

type dif site was 2.4 X 10-2, and this was reduced to 2.4 x 10-5when dif was deleted (A2336; Table 2). The efficiency ofintegration returned to the wild-type level when the straincontained the 33-bp insertion (dif3044). Surprisingly, the 33-bpinsertions at kb 1482 also restored a high level of recombina-tion (dif3185 and dif3186). The efficiency of integration intodif3186 was actually higher than that observed in the wild-typestrain. These experiments have also been conducted with acontrol plasmid which lacks the 33-bp sequence (pMAK705),and the frequency of integration was <10-5.

Double-dif strains were constructed to determine whetherdif3185 or dif3186 recombined with the wild-type dif+ site. InPK3343, the two sites (dif+ and dif3186) were present as adirect repeat, and deletion of the intervening 118 kb of DNAwas readily observed by the loss of the gene for formatedehydrogenase (20). Southern hybridizations of one of thesederivatives (PK3303) demonstrated that it contained a fusionfragment of the expected size, formed by recombinationbetween the two dif sites (Fig. 2, lanes 6 and 10). Thesederivatives had a wild-type phenotype with respect to filamen-tation (data not shown) and SOS induction (Table 1). Thefrequency of excision of the 1f8-kb region was - 1% pergeneration and was dependent on both XerC and XerD.

Strain PK3302 contained the two dif sites as an invertedrepeat (dif+ and dif3185), and recombination would be ex-pected to invert the intervening DNA. InxerC+ cells grown for50 generations, the two original dif sites as well as two newhybrid sites were present at equal frequency (Fig. 2, lanes 5 and9). This demonstrates that inversion occurred, but there was noselective advantage for either orientation.We tested whether recombination occurs between the 33-bp

sites at kb 1482 (dif3186) and kb 1600 (dif3044) in PK3545.Derivatives in which the intervening DNA had been deletedwere recovered, but the frequency was at least 100-fold lowerthan that observed between dif+ and dif3186. This low fre-quency made it difficult to determine whether the recombi-

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Table 1. sfiA::lacZ expression

,3-Galactosidase activity*

- Casamino + CasaminoStrain dif allele acids acids

2649 dif+ (4 dif+) 145 1062650 A2336 (12-kb deletion) 623 11643140 A2035 (155-kb deletion) 2283 14683044 dif3044 ( 33 bp in 12-kb deletion) 121 2243547 diJ3547 ( 33 bp in 173-kb deletion) 102 1563280 dif3185 (33 bp -- and A2336) 707 12553281 dif3186 (4- 33 bp and A2336) 748 11983303 dif3303 (recombinant between <- dif3186 and dif+) 187 1243546 dif3546 (recombinant between +- dif3186 and dif3044) 175 269

Cells were grown in M9 glycerol medium with or without 0.5% Casamino acids.*Miller units (12).

nation required XerC and XerD or whether homologousrecombination occurred in the 1.3-kb spc genes, which wouldbe present as a direct repeat. Regardless, the low frequencyindicates that XerC/XerD-mediated recombination was se-verely reduced in this strain. Strains carrying this hybrid site(dif3546) exhibited a few filamentous cells (Fig. 3D), and thelevel of SOS induction, although low, was higher than in theother suppressed strains in medium supplemented withCasamino acids (Table 1).

DISCUSSIONThe experiments described here demonstrate that the Difphenotype, caused by a deletion of the dif site and either 12 or173 kb of flanking DNA, can be suppressed by insertion of a33-bp DNA fragment at the site of the deletion (dif3044 anddif3547; Fig. 1). Suppression was determined by the absence offilamentation (Fig. 2) and the level ofSOS induction (Table 1).This latter procedure showed that although suppression wascomplete in cells growing in glycerol minimal medium, SOSwas still slightly induced in more rapidly growing cells. Thiscould be a result of the increased origin/terminus genefrequency ratio expected in these conditions. For example, theaddition of Casamino acids decreased the doubling time ofPK3044 from 103 to 56 min, which would increase theorigin/terminus ratio -25% (1). Increased interactions be-tween homologous regions could increase the frequency ofsister chromatid exchange, which would in turn increase theneed for dimer resolution.

Since suppression occurred even though 12 or 173 kb hadbeen removed from the terminus, the 33-bp sequence does notrequire any specific flanking region in order to function. Ifother site-specific proteins are required at dif, binding wouldhave to be in the same region that binds XerC and XerD. TheXerC-XerD-dif complex itself might also provide a bindingsite for additional proteins. It should be noted that the 173-kbdeletion removed TerB, TerC, and the tus gene (9), as well assix of the "hotspots" for homologous recombination (21). Thismight remove an alternative resolution pathway and be thebasis of the more extensive SOS induction due to A2035 (Table1). Since suppression due to the 33-bp sequence was notcomplete in all conditions, other factors have some effect on

Table 2. Integration efficiency of pPK706 at various sites

Strain dif allele Integration efficiency*2649 recA dif+ 2.4 x 10-22650 recA A2336 2.4 x 10-53044 recA dif3044 2.1 X 10-23280 recA dif3185 A2336 4.3 x 10-33281 recA dif3186 A2336 6.5 x 10-2

*No. Cmr at 42°C/no. Cmr at 28°C.

recombination at dif But the degree of suppression attained inspite of the 173-kb deletion indicates that it should be possibleto suppress the phenotype of even larger deletions, such asA1608, which removed 350 kb of DNA from the terminusregion (22).The absence of a requirement for flanking sites illustrates an

important difference between resolution at the difand cer sites.cer is a topologically restrained system, and it contains a 200-bpflanking region which binds ArgR and PepA (23). cer-mediated dimer resolution is at least 104 times more frequentthan dimer formation (24), and this directionality requires theflanking sequences and auxiliary proteins. It has been pro-posed that these sites facilitate formation of an intra dimercomplex in the small ColEl plasmid, and recombination occurspreferentially at these sites. Since the large size of the bacterialchromosome probably eliminates any capacity for sites indimers to be distinguished from those in monomers (10),accessory sites and proteins of this type would be useless. Asimple alternative model for resolution at dif proposes thatdirectionality is provided by partitioning itself. The dif siteswould recombine randomly after they were replicated, irre-spective ofwhether they were present in monomers or a dimer.The ongoing segregation of the daughter chromosomes wouldeventually place the sites in monomers that were sufficientlyseparated so that no more interaction would occur.Although the Dif phenotype can be suppressed by insertion

of a 33-bp sequence, the location of the insertion is quiteimportant. There was no suppression when this sequence wasinserted in either orientation at kb 1482 (dif3185 and dif3186;Fig. 1). This was surprising, since the site was in the terminusregion and only 118 kb from the normal dif site. Furthermore,these insertions still functioned as RecA-independent recom-bination sites (Table 2). This indicates that the requirementsfor function as a chromosomal resolvase site are more de-manding than those for recombination with a plasmid.

Further studies of dif3185 and dif3186 indicated that theycould recombine with the wild-type dif+ site, when present indouble-dif strains. Sites in inverted orientation (dif+ anddif3185) inverted the intervening DNA (Fig. 2, lanes 5 and 9).Sites in direct orientation (dif+ and dif3186) deleted the inter-vening DNA (Fig. 2, lanes 6 and 10). This formed a hybrid sitewhich lacked 118 kb of DNA on the left-hand side of the 33-bpsequence but which contained DNA normally present on theright-hand side (dij3303; Fig. 1). The Dif phenotype was sup-pressed in these strains.

dif3186 also recombined with the 33-bp site present at thenormal dif location (dif3044). The frequency of this deletionwas reduced -100-fold compared with deletions at the wild-type site. The absence of a complete difsite apparently affectedsome step in deletion formation. The resulting difsite (dif3546)was functional, although some SOS induction still occurred(Table 1; Fig. 3D). This dif site harbored a 118-kb deletion on

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the left-hand side and also had a 3-kb deletion on the right-hand side of the 33-bp sequence (Fig. 1).One possible explanation for the regain of function by

dif3186, following deletion of DNA between kb 1482 andwild-type dif or dif3044, is that this region contains a site thatinhibits function of ectopic dif sites. If such a site is present, itwould have to be present between kb 1482 and kb 1544.Combination of dif3186 with A2035 (kb 1544-1695) or A2038(kb 1614-1698) did not restore suppression. Based on prelim-inary studies with other insertions (unpublished experiments),removal of a single inhibiting locus is not sufficient to explainthe regain of function by ectopic dif sites.

It is unclear why dif functions only in a limited part of theterminus region. This could be due to chromosome structure,and the structure of the terminus region enhances interactionsat dif When dif is moved to an ectopic position, this possiblyplaces it such that interactions between sites in newly formeddaughter chromosomes are restricted. A simple basis forsequestration could be that processing of newly synthesizedDNA into separate nucleoids occurs shortly after replication(3). The normal location of difcould be in a region that delaysthis processing and maximizes interactions. Nonsuppressingsites could still interact with dif-containing plasmids if theplasmids were not sequestered into nucleoids. Also, plasmidswould have a higher copy number, and interactions couldprobably occur throughout the cell cycle.The observation that dif functions only in certain locations

provides a means to study the structure of this region of thechromosome. Recombination at dif sites can be used tocharacterize the various types of DNA interactions that occur.Suppression provides the most sensitive assay, but it will alsobe important to determine the frequency of deletion andinversion formation and of integration of dif-containing plas-mids. This information can be combined with that obtainedfrom other recombination assays, which also demonstratedunusual phenomena in the terminus. Louarn et al. (25) havenoted that the frequency of recombination is greatly elevatedin this region, and Horiuchi et al. (21) have identified "hot"DNA sites, which are highly recombinogenic X sites.

This research was supported by National Institutes of Health GrantGM32968.

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