Proc. Natl. Acad. Sci. USAVol. 86, pp. 3489-3493, May 1989Biochemistry

Vaccinia DNA topoisomerase I promotes illegitimate recombinationin Escherichia coli

(bacteriophage A/lysogenic induction/prophage excision)

STEWART SHUMANProgram in Molecular Biology, Sloan-Kettering Institute, New York, NY 10021

Communicated by Jerard Hurwitz, January 10, 1989

ABSTRACT Vaccinia virus encapsidates a M, 32,000 typeI DNA topoisomerase. Although the vaccinia gene encoding thetopoisomerase is essential for virus growth, the role of theenzyme in vivo remains unclear. In the present study, thephysiologic consequences of vaccinia topoisomerase action havebeen examined in a heterologous system, Escherichia coli. Thevaccinia topoisomerase gene was inducibly expressed in an intFA lysogen BL21(DE3) using a T7 RNA polymerase-basedtranscription system. Expression of active topoisomerase in thiscontext resulted in recA-dependent lysogenic induction as wellas cell lysis. Surprisingly, topoisomerasv expression also ef-fected a 200-fold increase in the titer of infectious A phage,apparently by promoting int-independent prophage excision.This effect was not observed during lysogenic induction withnalidixic acid. Restriction analysis of genomic DNA fromplaque-purified excisants revealed (in 10 of 10 cases) grossalterations of the DNA structure around the att site relative tothe structure of the parental phage DE3. It is construedtherefore that vaccinia DNA topoisomerase I acts to promoteillegitimate recombination in E. coli.

Eukaryotes possess two classes of DNA topoisomerases,types I and II, that differ in their mechanisms of strandcleavage, cofactor requirements, and sensitivities to drugs (1,2). A role for the topoisomerases in such key cellular eventsas DNA replication, chromosome segregation, genetic re-combination, and transcription has been suggested. Geneticexperiments in yeast have evinced an essential function forDNA topoisomerase II in cell growth (3, 4); topoisomerase I,on the other hand, is dispensable for yeast viability (3-5).Thus, the assignment of a particular role to eukaryotictopoisomerase I is less than straightforward and appears to becomplicated by the redundancy of function of the types I andII enzymes. For example, yeast top] mutants that lack thetype I enzyme have no readily discernable phenotype but dohave profound defects in macromolecular synthesis in thecontext of superimposed mutations in the gene encoding typeII topoisomerase (6).A eukaryotic viral system, vaccinia, provides a useful model

for the study of type I topoisomerase. Vaccinia, a cytoplas-mically replicating poxvirus, contains within the infectiousvirion a type I DNA topoisomerase (7). The vaccinia enzymeis mechanistically similar to cellular type I enzymes but isdistinguished by its smaller size, Mr 32,000 (8, 9). The virusencapsidated enzyme is also encoded by the vaccinia genome(8, 10). The topoisomerase structural gene specifies a poly-peptide of 314 amino acids that includes a region of sequencesimilarity to the type I topoisomerase of yeast (5, 8). Inser-tional mutagenesis studies suggest that the topoisomerasegene is essential for virus growth in cell culture (11).

Analysis of the effects on E. coli of the expression ofeukaryotic type I topoisomerases may well provide insightsinto the functional capabilities of these enzymes in vivo,albeit somewhat out of their natural context. Bjornsti andWang have shown that expression of yeast topoisomerase Iin a temperature-sensitive topA strain of Escherichia coliconfers the ability to grow at the nonpermissive temperature(12). This genetic complementation assay thus provides aphenotypic screen for topoisomerase I that is lacking in yeastitself. In the same vein, it was of interest to determine whateffects vaccinia topoisomerase I expression would have onbacterial physiology. The expression strategy involved clon-ing the topoisomerase gene into a plasmid so as to place itunder the control of a bacteriophage T7 promoter. Thisplasmid was then used to transform a A lysogen of E. coli thatcontains an inducible T7 RNA polymerase gene inserted intothe A int gene. I report here that expression of vacciniatopoisomerase in this context leads to recA-dependent lyso-genic induction, as well as int-independent prophage exci-sion. A possible role for the eukaryotic topoisomerase I inrecombination is suggested.

METHODS

Bacteria and Plasmids. The E. coli strains used for topo-isomerase expression, referred to as BL21(DE3) and HMS174-(DE3), are A DE3 lysogens ofhost strains BL21 (F-, hsdS, gal)and HMS174 (F-, hsdR, recA, RifR). DE3 (imm2l) containsthe T7 RNA polymerase gene under the control of a lacUV5promoter, inserted within the int gene of the phage (13).Additional derivatives of these lysogens contain the plasmidpLysS or pLysE. The pLys plasmids, which confer chloram-phenicol resistance, contain a fragment of T7 DNA thatincludes the T7 lysozyme gene inserted into pACYC184.pLysE and pLysS differ only in the orientation of the lyso-zyme gene with respect to the tet promoter, such that higherlevels of lysozyme expression are achieved with pLysE. T7lysozyme is a specific inhibitor of T7 RNA polymerase (14)and is useful in expression studies in lowering the basal activityof T7 RNA polymerase in the DE3 lysogens. PlasmidspA9topo and pC6topo contain the vaccinia topoisomerasegene inserted into the expression vector pAR3038 (15); in thecase of pA9topo, the gene is in the sense orientation withrespect to the T7 promoter, while in pC6topo, the vacciniagene is in the antisense orientation. Previous studies hadshown that pA9topo, but not pC6topo, could program thesynthesis of active vaccinia topoisomerase in E. coli BL21upon provision ofT7RNA polymerase by phage infection (10).These plasmids, which confer ampicillin resistance, were usedto transform BL21(DE3), HMS174(DE3), and related pLys-containing strains.

Abbreviation: IPTG, isopropyl P-D-thiogalactopyranoside.

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RESULTS

Expression of Vaccinia Topoisomerase in ADE3 Lysogens.Vaccinia topoisomerase can be expressed in active form in E.coli under the control of a T7 promoter by using A infectionto deliver T7 RNA polymerase to the cell (10). This method,which does not allow for fine control of the amount of targetgene expression and which also results in killing of theinfected cell, is not suitable for assessing the effects on E. coliof vaccinia topoisomerase expression. Thus, to address thispoint, I turned to the inducible lysogen-based expressionsystem described by Studier and colleagues (13).A culture of BL21(DE3)pLysEpA9topo was induced to

express T7 RNA polymerase by the addition of 0.4 mMIPTG. Upon induction, the concentration of newly made T7RNA polymerase can be expected to exceed that of theinhibiting lysozyme and thus drive the expression of vacciniatopoisomerase. The growth characteristics of the inducedculture are shown in Table 1. An initial period of exponentialgrowth was followed by lysis of the bacteria at 1.5-2 hrpostinduction. Lysis required the expression of the vacciniatopoisomerase gene, since otherwise identical cells carryingthe antisense topoisomerase expression plasmid pC6topo didnot undergo lysis. The dramatic effects of IPTG on cellgrowth correlated with the appearance of active vacciniatopoisomerase, as shown by assay of cell lysates for EDTA-resistant DNA relaxing activity (Fig. 1). Three distinct colonyisolates of BL21(DE3)pLysEpA9topo displayed a markedincrease in relaxing activity postinduction. Cells bearingpC6topo manifested no increase postinduction.

Topoisomerase Expression Causes Lysogenic Induction.How might vaccinia topoisomerase cause lysis of the hostbacterium? Topoisomerase expression could lead to lyso-genic induction of the prophage, resulting in late viral geneexpression and concomitant cell lysis by either the A-encodedor the pLysE plasmid-encoded lysozyme. Lysogenic induc-tion depends on cleavage of the A repressor by recA protein(19); thus, the role of lysogenic induction in topoisomerase-

Table 1. Induction with IPTGA600/Aphage titer, pfu/ml

Time, BL21(DE3)pLysEhr pA9topo

BL21(DE3)pLysEpC6topo

Experiment 10 0.256/190 0.267/100.5 0.480/450 0.414/201.0 0.740/460 0.576/201.5 0.184/21,000 0.810/<102.0 0.054*/40,000 0.956/20

Experiment 2HMS174(DE3)pLysE HMS174(DE3)pLysE HMS174(DE3)-

pA9topo pC6topo pLysE

0 0.186/<10 0.254/<10 0.227/<100.5 0.237/<10 0.323/<10 0.286/<101.0 0.299/<10 0.379/<10 0.354/<101.5 0.322/<10 0.504/<10 0.5151<102.0 0.326/<10 0.725/<10 0.688/<102.5 0.353/<10 0.853/<10 0.883/10Bacteria were grown in LB medium. Ampicillin (100 jug/ml)

and/or chloramphenicol (30 Ag/ml) were included as needed tomaintain the plasmids. Logarithmically growing cells (A600 0.2-0.3)were induced to express T7 RNA polymerase by addition of IPTG to0.4 mM concentration at time 0. Further cell growth was followed atthe indicated intervals by measurement of A6Eo. To determine thephage titer of the cultures, aliquots were withdrawn, made 2% inCHCl3, and centrifuged to remove bacteria and cell debris. Serialdilutions of the supernatants were titered on BL21 plating bacteria asdescribed (17). pfu, Plaque-forming units.*Lysis of cultures.

CI

*- S

FIG. 1. Induction of vaccinia DNA topoisomerase by IPTG.Cultures (10 ml; A6w 0.4-0.6) of BL21pLysE transformants contain-ing the topoisomerase expression plasmid in the sense (pA9-8, pA9-9,and pA9-2) or antisense (pC6-6) orientation were made 0.4 mM inIPTG and allowed to grow for an additional 2 hr. A parallel set ofcultures received no IPTG. Cells were pelleted and resuspended in 1ml of 150 mM NaCl/50 mM Tris HCI, pf4 7.5/10 mM EDTA/1 mMdithiothreitol/10% (wt/vol) sucrose. Lysis was achieved by twocycles of freezing and thawing with subsequent addition of NonidetP-40 to 0.01%. The lysates were clarified by centrifugation at 100,000x g for 60 min in a type 60 Ti rotor. The supernatant was removed andassayed for protein concentration (18) and EDTA-resistant topoisom-erase activity. Topoisomerase assays were performed as described (8)using pBR322 DNA as substrate, activity being gauged by the con-version of superhelical DNA to the relaxed form. Each reactionmixture contained 100 ng of lysate protein; this was done to correct forthe uniformly lower concentration of protein in IPTG-inducedpA9topo lysates owing to the extent of cell lysis prior to harvesting thebacteria. Reaction products were analyzed by agarose gel electropho-resis (8); a photograph of the gel is shown. The symbols above eachlane denote the bacterial isolate used as a source of enzyme. Indudedcultures are indicated by a plus sign; uninduced cultures are indicatedby a minus sign; C, control reaction from which enzyme was omitted.The positions of supercoiled (S) and relaxed (R) forms of DNA areindicated by arrows.

dependent lysis could be tested by performing similar exper-iments in a recA strain, HMS174. As shown in Table 1,topoisomerase expression in this background caused arrest ofgrowth at 1.5 hr postinduction but no discemable cell lysis.Arrest of growth was attributable to vaccinia topoisomerase,since neither the HMS174(DE3) lysogen itself nor the lysogencarrying the antisense topo plasmid pC6topo experienced asimilar cessation of growth.Topoisomerase-Dependent Prophage Excision. It was antic-

ipated that cell lysis would not be accompanied by thegeneration of progeny phage, because of the int- genotype ofthe integrated DE3 prophage. It was surprising, therefore, tonote that IPTG induction of topoisomerase expression re-sulted in a 200-fold increase in the infectious phage titer(Table 1). This phage burst was not evident in the strainbearing pC6topo. This increase in A titer, like lysis of thecells, required both topoisomerase expression and a wild-type recA protein.To explore further the basis of cell lysis and of the phage

burst, a parallel series of experiments was performed withnalidixic acid, an inhibitor of DNA gyrase and a classicalinducer of recA, to effect lysogenic induction of the samebacterial strains. As shown in Table 2, exposure of growingcells to nalidixic acid resulted in synchronous lysis ofBL21(DE3)pLysE strains with a time course similar to that oftopoisomerase-dependent lysis by IPTG. Nalidixic acid, un-like IPTG, caused lysis whether or not a T7 target plasmidwas present. Also, as expected, nalidixic acid induced lysisonly in a recA+ background. The depressed rate of growth ofthose strains not lysed (Table 2) was consistent with theinhibitory effects of the drug on bacterial DNA replication. Inaddition, we can infer from the failure of nalidixic acid to lyseBL21(DE3) lacking a pLys plasmid that it is the accumulatedT7 lysozyme, rather than the A lysozyme (gene R product),that is responsible for lysis in this case, as well as in thetopoisomerase induction experiments.

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Table 2. Induction with nalidixic acid

A6M/A phage titer, pfu/mlTime, BL21(DE3)pLysE BL21(DE3)pLysE BL21(DE3)-hr pA9topo pC6topo pLysE

Experiment 10 0.225/70 0.265/70 0.245/700.5 0.340/70 0.425/50 0.426/1101.0 0.390/110 0.516/20 0.491/701.5 0.139/60 0.312/10 0.161/1102.0 0.052*/80 0.089*/10 0.056*/2002.5 0.052*/80 0.076/<10 0.063*/140

Experiment 2BL21(DE3)pLysE HMS174(DE3)pLysE BL21(DE3)

0 0.257/70 0.226/<10 0.256/<100.5 0.380/70 0.280/<10 0.414/<101.0 0.456/30 0.341/<10 0.501/<101.5 0.326/40 0.449/<10 0.566/<102.0 0.086*/10 0.509/<10 0.554/<102.5 0.057*/10 0.579/<10 0.567/<10Logarithmically growing lysogens were induced by addition of

nalidixic acid to 50,ug/ml at time 0. Further cell growth was followedat the indicated intervals by measurement of A6Eo. The phage titer ofthe cultures was determined as described in Table 1. pfu, Plaque-forming units.*Lysis of cultures.

Comparison of the phage titers after nalidixic acid-mediated lysogenic induction with those after topoisomerase-mediated induction revealed striking differences. In none ofthe strains tested did nalidixic acid cause an increase in phagetiter. This is reflective of the lack of int function required forexcisional recombination of the prophage and confirms thetightness of the into phenotype in the DE3 lysogens. Thus,the phage burst noted in Table 1 must be due to topo-isomerase-dependent int-independent excision of the pro-phage. The burst cannot be merely a function of cell lysis(i.e., with release of preexisting intracellular phage particles)since the lysis of pA9topo-bearing BL21(DE3)pLysE bynalidixic acid caused no change in phage titer. The magnitudeof the phage yield (on the order of lo- plaque-forming unitsper cell) is significantly lower than that expected fromexcision of wild-type prophage. We cannot readily distin-guish whether this is due to a low efficiency of topo-isomerase-dependent excision, or if the true potential forphage production is underestimated by the relatively rapidonset of cell lysis to T7 lysozyme.

Analysis ofA Excisants. An outstanding question is whethervaccinia topoisomerase-dependent excisional recombinationis site specific or illegitimate. If vaccinia topoisomerase istruly acting in place of int in a physiologic manner, it is to beexpected that recombination would occur between attL andattR to regenerate attP in progeny phage (20), as depicted inFig. 2. If recombination is illegitimate, progeny phage willhave altered DNA sequences at the att locus. This issue wasaddressed by plaque-purifying phage isolates from IPTG-induced BL21(DE3)pLysEpA9topo, amplifying the ex-cisants, and then analyzing the structures of their att sites.The phage DNA of 10 such isolates was digested with severalrestriction endonucleases, and the digests were compared tothose of the parental DE3 phage. The restriction map of DE3(13, 21) and the expected sizes of DNA fragments afternuclease digestion are shown in Fig. 2; the results of therestriction analysis of the parental and excisant A DNAs areshown in Fig. 3.EcoRI digests of the topoisomerase-induced excisants re-

semble those of the parent DE3 except with respect to the10.1-kilobase (kb) restriction fragment that spans attP (Fig.3A). This region of the genome has been permuted in severalcases to yield single EcoRI fragments that are either longer

(a) X E

cosLE HB B E E E' "i 4 4 A a cosR

l - 21.5 10.1 - 5.0|53..1--3.5-

Eco Rl21 53110111497631003500

EcoRl/Hindil121531

1 3738738497631003500

(b) DE3 ProphageB p E

i

nILL

BamHI23397441815403

EcoRI/BamHI21 531

1 86644183827497631 003500

E Hl Mt% -.1vlv

(c) Prophage Excision

B B EattL;i i

3 '"% I 7-.O 1 ,10

E ' H Br @ 4,6,8,9L ',E H B I

I

*

attR

FIG. 2. Structure of A DE3 DNA in phage, prophage, andexcisant forms. (a) Structure of the genomic DNA of A DE3 isillustrated. DE3 is a derivative of A D69 (21) containing a 4.4-kbexpression cassette for T7 gene 1 inserted into the unique BamHIcloning site within the int gene (13). Solid line represents DNAderived from D69; hatched line represents the T7 cassette. The leftand right cohesive ends of the phage DNA are indicated by cosL andcosR. Restriction sites in DE3 for endonucleases EcoRl (E), HindI11(H), and BamHI (B) are marked by arrows under the appropriateletter. The location of the attP site is indicated by an arrow. Theborders and sizes of the EcoRI fragments (kbp) are depicted imme-diately below the genome. The sizes of restriction fragments (bp)expected from various endonuclease treatments are shown in tabularform. The fragments are listed in order of their position in the phageDNA, going from left to right. (b) Structure of the DE3 prophagepredicted from site-specific recombination between attP and attB.The recombinant junctions are indicated as attL and attR, respec-tively. The positions of restriction sites are marked by arrows.Internal regions of the prophage genome are indicated by the serratedline. Bacterial DNA is depicted as a thin line. (c) The sequencesaround attL and attR are approximated to illustrate the crossoverevent in prophage excision. Int-dependent excision normally in-volves recombination between attL and attR to reconstitute thephage genome shown in a. Topoisomerase-dependent recombinationoccurs at different sites around att (see text and Fig. 3). The roughlocations of the crossover points (relative to restriction sites) thataccount for the restriction patterns of the 10 excisants (Fig. 3) areshown as connecting lines. Restriction sites in A DNA are indicatedby arrows. Predicted sites for BamHI and for EcoRI cleavage withinthe bacterial DNA flanking attR are indicated by the arrows belowitalicized letters.

(isolates 1 and 2) or shorter (isolates 3, 5, 7, and 10) than theparental att-containing fragment. In the remaining instances,a new EcoRI fragment (of -2.5 kb in the case of isolates 4,6, 8, and 9, and -1.2 kb in isolate 7) is generated, along witha fragment of -7 kb. These data indicate clearly that topo-isomerase-dependent prophage excision does not reconsti-tute the wild-type att site.To localize approximately the crossover points for topo-

isomerase-dependent excisional recombination in the prog-eny phage, the DNAs were digested with endonucleasesHindIII or BamHI. HindIl cleaves uniquely in DE3 at a site

lbp"pz? - v

llve~

Biochemistry: Shuman

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Restriction Endonuclease Analysis of Lambda Excisants

><E'P B13 LcoRi HircIll

;? _ i. I ;-- - ix'_I I. "I 1

Cv BcarihI1MI p .6L 7!i D 7 F8 1 0

l:.~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

M P C i 2 3 4 5 6 7 8 9 1 0

) cE 3(0i1.- !TI H1

rM P J" 2 3 4 D6 8 9 1 0

FIG. 3. Restriction endonuclease analysis of A DNAs. Twoisolates of the parental DE3 virus (designated P and P') and 10excisant progeny from an IPTG-induced culture of BL21pLysEpA9-topo (numbered 1-10) were plaque-purified and amplified in BL21 asdescribed (13), first by growth in a 35-ml culture followed by growthin a 1-liter culture until lysis was complete. Phage was collected bypolyethylene glycol precipitation and rapid isopycnic banding inCsCl. A DNA was isolated as described (17). Restriction digests withthe indicated endonucleases were performed in 20-1.l reaction mix-tures. After 2-3 hr of incubation, the restriction products wereanalyzed by electrophoresis through 0.7% agarose gels containingTBE (90 mM Tris/64.6 mM boric acid/2.5 mM EDTA, pH 8.3) and0.5 gg of ethidium bromide per ml. Photographs of the gels areshown. The source of A DNA in each reaction mixture is indicatedabove the lane. LaneM contains aHindIII digest of wild-type A DNAthat was used as size marker in this analysis. The sizes of the markerfragments (in bp), in order of increasing mobility, are 23,130, 9416,6557, 4361, 2322, and 2027.

within the 10.1-kb EcoRI fragment located 252 base pairs (bp)to the left of attP. Digestion with EcoRI and HindIII can beexpected to yield a 1.37-kb fragment from the left end and an

8.7-kb fragment from the right end of the att-containingEcoRI fragment of DE3 (Fig. 3B). All excisants exceptexcisant 3 retain the HindIII site in the genomic DNA, as

evinced by the liberation of 1.37-kb fragment with a concom-itant decrease in the size of the att-containing fragment.Thus, the crossover points in these cases must lie to the rightof the HindIII site. Excisant 3 has lost the HindIII site (notethe lack of shift in mobility of the att-containing fragment),thereby localizing the crossover point to the region betweenthe EcoRI site and the HindIll site.BamHI cleaves DE3 at two sites flanking the 4.4-kb T7

insert in the int gene; the leftward BamHI site is 241 bp to theright of attP. Many of the excisants (nos. 3, 4, 6, 8, and 9)have deleted one of the BamHI sites, as manifest by the lackof a 4.4-kb BamHI fragment and the presence of only twolarge BamHI fragments (Fig. 3C). Other excisants (nos. 2, 5,and 10) appear to have substituted aBamHI site for one ofthe

parental sites, as shown by the appearance of internalBamHIfragments of novel size. Combined digestion withBamHI andEcoRI localizes theBamHI sites within the excisionjunction-containing EcoRI fragment. As shown in Fig. 3D, BamHIliberates from DE3 a 1.8-kb fragment from the left end of theEcoRI fragment, a central 4.4-kb T7 cassette, and a 3.8-kbfragment from the right end of the EcoRI fragment. In theexcisants, the novel fragments generated by either enzymealohe (e.g., the 2.5- and 1.2-kb EcoRI fragments in Fig. 3A,and the 5.4- and 1.8-kb BamHI fragments in Fig. 3C) are notaltered by treatment with the other enzyme. In 9 of 10 cases(no. 3 excepted), the leftward EcoRI/BamHI fragment hasbeen replaced with a fragment of -2.2 kb. The rightward3.8-kb BamHI/EcoRI fragment has been preserved in iso-lates 1, 2, 3, 5, and 10 but is replaced in isolates 4, 6, 7, 8, and9 with a fragment of -5.2 kb. The restriction patterns can beaccounted for by postulating in almost all cases (except no.3) that excision arose by crossing over between bacterialsequences upstream of attR and phage sequences lyingbetween the leftwardBamHI site and the EcoRI site (Fig. 2c).To account for the creation of novel restriction fragments, weposit the presence of a BamHI site in the bacterial DNAflanking attR (located 0.8 kb to the right of the A HindIII site)and an EcoRI site flanking attR (located 5.2 kb to the right ofthe BamHI site; see Fig. 2c). These postulated restrictionsites have indeed been reported (ref. 22 and referencestherein). The exceptional case is phage 3, which has appar-ently recombined among sequences in the leftward EcoRI/HindIII fragment and sequences in the BamHI/BamHI frag-ment of DE3. The proposed crossovers are illustrated in Fig.2c. The essential point of this analysis is that in no case wasexcision accomplished by site-specific recombination be-tween attL and attR; thus, topoisomerase I-dependent re-combination is apparently illegitimate.

DISCUSSIONExcision of an integrated A prophage from the E. coli chro-mosome occurs via site-specific recombination between attLand attR sequences (reviewed in ref. 20). This reactioninvolves two phage-encoded proteins, the products of the intand xis genes, respectively, and a host protein, IHF, con-sisting of two subunits encoded by the E. coli himA and hipgenes (26). The requirement for IHF and xis proteins inexcisive recombination is not absolute in that mutationsaffecting the int gene can relieve the strict need for the otherproteins (27). Int protein, on the other hand, is absolutelyrequired for site-specific excision. The most remarkablefinding of the present study is that vaccinia topoisomerasepromotes prophage excision in the absence of functional intprotein. This is reflected in the 200-fold increase in infectiousphage titer after IPTG treatment of topoisomerase-ex-pressing lysogens. The magnitude of this effect is even

greater (2000-fold) when comparison is made between topoi-somerase-containing and non-topoisomerase-containinglysogens (Table 1). Therefore, the eukaryotic topoisomeraseI is capable of complementing int function in vivo. The intprotein, a Mr 40,000 polypeptide, is itself a type I DNAtopoisomerase that can relax positive as well as negativesupercoils in vitro without obvious sequence specificity (28).Int-catalyzed DNA relaxation proceeds via a covalent DNA-protein intermediate involving a 3' phosphoryl linkage totyrosine 342 of the int protein (29, 30). Int topoisomerase isthus similar in its biochemical properties to the family ofeukaryotic type I enzymes that includes vaccinia topoisom-erase. The topoisomerase activity of int protein is requiredfor int action as a recombinase (30).

It is inferred from the present results that the vacciniatopoisomerase is capable of promoting excisive recombina-tion in E. coli. Topoisomerase-dependent prophage excision

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appears to occur via illegitimate, rather than site-specific,recombination and is thereby distinguished from excisiverecombination catalyzed by int protein. The specificity of intrecombinase for the att sites is a function of binding of theprotein to specific DNA sequences within the att regions (20).The degree of specificity, if any, of the vaccinia topo-isomerase-mediated recombination will be revealed by finermapping and sequencing of the recombination junctions. Atfirst glance, however, the process would seem to be nonran-dom, insofar as: (i) 9/10 of the junctions lie to the right of theHindIII site in DE3, and (ii) several of the excisants haveremarkably similar restriction patterns. Whether these ob-servations are due to bias inherent in the assay (i.e., in somepeculiarity of DE3 that selects for viable progeny that haverecombined in a particular manner) or to the specificity oftopoisomerase-mediated strand cleavage remains to be de-termined. While the former model cannot be dismissed, thenonessential nature of the genomic region surrounding attP ineither direction makes this alternative less likely.

Previous studies have implicated the DNA topoisomerasesin illegitimate recombination. E. coli DNA gyrase and bac-teriophage T4 topoisomerase (both type II enzymes) as wellas calf thymus topoisomerase II can promote illegitimaterecombination in vitro (31-33). A possible role for the eu-karyotic type I topoisomerase in illegitimate recombinationhas also been invoked (34). The latter proposal is sustainedby the finding that nucleotide sequences surrounding ex-change sites for illegitimate recombination in vivo correlatewith the preferred recognition sequences of topoisomerase Iin vitro. The present study provides compelling support forthe view that a eukaryotic topoisomerase I can promoteillegitimate recombination in vivo by demonstrating the pos-itive relationship between the presence of topoisomerase Iactivity and the occurrence of illegitimate recombination.The in vivo assay for recombination-the generation of in-fectious excisant phage-while extremely sensitive, may wellunderestimate the overall extent of recombination events,either in remote areas of the chromosome, or in regions of theprophage that reconstitute noninfectious phage.

It is not clear from this work whether vaccinia topoisom-erase per se is sufficient to mediate recombination or whetherbacterial components contribute to the effects presently re-ported. In this regard, relaxation of the bacterial chromosome(a probable sequela to the expression of vaccinia topoisom-erase in E. coli) has been shown to increase the synthesis ofDNA gyrase (35). Gyrase could then be the agent of illegiti-mate prophage excision, with vaccinia topoisomerase playingonly an indirect role. If this were the case, however, it isexpected that prophage excision would also be augmented bynalidixic acid, since illegitimate recombination catalyzed invitro by prokaryotic type II topoisomerases is stimulated bythe quinolone drugs (16, 31, 32). Moreover, nalidixic acidinduces a 2- to 3-fold increase in the synthesis of the GyrA andGyrB subunits (35). The failure to observe an enhancement ofphage titer by nalidixic acid (Table 2) suggests that DNAgyrase is not responsible for vaccinia topoisomerase-dependent excision. The possible participation of other E. coliproteins in this process remains to be evaluated.

Finally, the observation that vaccinia topoisomerase par-ticipates in recombination in E. coli suggests a similar func-tion for this enzyme during vaccinia infection of eukaryoticcells. Indeed, vaccinia virus induces high levels of homolo-gous recombination in the cytoplasm of infected cells (23-25). The strand cleavage-rejoining activity of the topoisom-erase might be rendered specific for regions of DNA homol-ogy by interaction of the topoisomerase with some othervaccinia protein. Further clarification of the role of thetopoisomerase I in viral replication, and recombination in

particular, will be contingent on the isolation of conditionaltopoisomerase mutants and on the development of in vitrosystems for the study of viral recombination.

Some of the work reported herein was carried out in the Laboratoryof Viral Diseases, National Institutes of Health, Bethesda, MD, withthe generous support of Dr. Bernard Moss. Mr. Michael Golderprovided expert technical assistance. I thank Dr. F. W. Studier for hisgenerous gifts of bacterial strains, plasmids, and invaluable advice.Drs. Howard Nash, Michael Merchlinsky, Steven Broyles, PeterBullock, and Kenneth Marians provided insightful commentary.

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