Monomers and dimers of the RepA protein in plasmid pSC101 replication: domains in RepA

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Monomers and dimers of the RepA protein in plasmid pSC101 replication: domains in RepA

MANEN-COMMANDEUR, Danielle, UPEGUI-GONZALEZ, Lia Cristina, CARO, Lucien

Abstract

The replication of plasmid pSC101 requires the plasmid-encoded protein RepA. This protein has a double role: it binds to three directly repeated sequences in the pSC101 origin and promotes replication of the plasmid; it binds to two inversely repeated sequences in its promoter region and regulates its own transcription. A series of RepA protein derivatives carrying deletions of the C-terminal region were assayed for specific binding. We found that the last third of the protein is not needed for binding to the various specific sites. Truncated proteins that still bind can also form heterodimers with a wild-type protein. Analysis of band retardation assays conducted with wild-type and truncated proteins indicates that RepA binds to directly repeated sequences as a monomer and to inversely repeated sequences as a dimer.

MANEN-COMMANDEUR, Danielle, UPEGUI-GONZALEZ, Lia Cristina, CARO, Lucien.

Monomers and dimers of the RepA protein in plasmid pSC101 replication: domains in RepA.

Proceedings of the National Academy of Sciences, 1992, vol. 89, no. 19, p. 8923-8927

DOI : 10.1073/pnas.89.19.8923

Available at:

http://archive-ouverte.unige.ch/unige:138559

Disclaimer: layout of this document may differ from the published version.

Vol.89, pp. 8923-8927, October 1992 Microbiology

Monomers and dimers of the RepA protein in plasmid pSC101 replication: Domains in RepA

(DNAreplication/DNA-bindingproteins/binding sites)

DANIELLE MANEN, LIA-CRISTINA UPEGUI GONZALEZ*, AND LUCIEN CARO

Departmentof Molecular Biology,University of Geneva, 30 quai Ernest Ansermet, 1211 Geneva 4, Switzerland Communicatedby WernerArber,June 12, 1992 (receivedforreviewMarch 16, 1992)

ABSTRACT The replication of plasmid pSC101 requires theplasmid-encodedprotein RepA. This protein has a double role: it binds to threedirectly repeated sequences in the pSC101 origin and promotesreplication of the plasmid; it binds to two inversely repeated sequences in its promoter region and regu- lates its owntranscription. A series of RepA protein derivatives carrying deletionsoftheC-terminal region were assayedfor specific binding. We found that the last third of theprotein is notneeded for binding to the various specific sites. Truncated proteins that still bind can also form heterodimers with a wild-type protein. Analysis of band retardation assays con- ducted with wild-type and truncated proteins indicates that RepA binds todirectly repeated sequences as a monomer and toinversely repeated sequences as a dimer.

The replication of plasmid pSC101 in Escherichia coli de- pends on the plasmid-encoded protein RepA and on host- encodedproteins suchas IHF(1, 2) andDnaA(3,4). RepA has 316amino acids anda calculated molecularmassof37 kDa and is weakly basic with a net charge of +6 (5-9).

Interactions betweenRepA, IHF,andDnaA, bindingtothe replication origin,areinvolved informingstructuresrequired for theinitiation ofreplication (10). A 2.2-kilobaseHincII- RsaIfragment contains all the functions and sites essential forpSC101replicationincluding therepA gene(5-8, 11). The replication region (Fig. 1) contains an A+T-rich segment followed by three directly repeated sequences, RS1, RS2, andRS3. The RS1 andRS3 sequences differby 1 outof24 basepairs (bp); RS2 is shorter, with 18 homologous basepairs (Fig.2B). Apartially homologoussequence,RS4,isfoundto the right of RS3; it overlaps the leftarmof thepalindrome shownasIR1.TherepA promoterstraddlestwopalindromic (inverselyrepeated) sequences,IR1and IR2, whicharealso partially homologoustothedirectlyrepeatedsequences(ref.

12;Figs.1and 2B).In vitrotheRepAproteinappears tobind to all these sequences but more efficiently to the inversely repeated sequences thantothedirectly repeated sequences (10, 12-14). In vivo itautoregulates the transcription of its mRNA (15-17). Mutations in RepA or within the three repeated sequences upstream canabolishreplication (5, 18, 19). Thissuggests anessential role for thebindingofRepAto these sequences in theinitiation ofreplication. Thereplica- tion ofpSC101 has been reviewed recently (20).

Asafirst stepin analyzing thefunctional domains of the protein, deletions of the 3' region ofthe repA gene were produced. The truncated RepA proteins were assayed for DNAbinding.Wefound that the last third oftheC-terminal end isnotneededforspecificbindingtodirectlyrepeatedor inverselyrepeated sequences. Wealso foundevidence sug- gestingthatRepA bindsto directly repeated sequences and initiatesreplication in monomeric form but that it binds to

inversely repeated sequences and autoregulatesitstranscrip- tion indimeric form.

MATERIALS AND METHODS

Bacterial Strains and Plasmids. The bacterial strains used in this study were C600 (supE44 hsdR thi-J thr-J leuB6 lacYJ tonA21)andHB101[supE44hsdS20(rBmBi)recA13 ara-14 proA2 LacY) galK2 rpsL20xyl-5 mtl-i]. Subcloning of dif- ferentfragments of pSC101 (see below) was done by using plasmid pUC19 (21) orplasmidpBR322 (22).

DNA Methodology. Enzymes were purchased from com- mercial suppliers, and their recommended conditionswere employed for the reactions. Cloning procedures,transforma- tions inE.coli,plasmidDNApreparation, andDNAlabeling by the Klenow fragment of DNA polymerase were done accordingtoSambrooketal. (23).

Crude Protein Extracts. The proteins were extracted by sonicationaccordingtoBetermieretal. (24). Thequantity of total proteinwasmeasuredby the method ofBradford (25).

Gel Retardation. A 20-plI binding reaction mixture con- sisted of '15

Ag

Aul

bovine serum albumin (0.25 mg/ml)/0.5 mM dithiothrei- tol/25mMMgCl2/1 MNaCl], and5 ngof32P-labeled DNA fragment. Binding was carried out at 16°C for20 min. The boundcomplexwasanalyzed ina7.5%acrylamidegel.After beingrun at 150Vfor2h, thegelwas dried and autoradio- graphed.

PlasmidConstructions. ThepSC101coordinatesarethose usedby Churchwardetal. (6). Forcreating deletionsatthe 3' end,weusedexisting restriction sites (HaeIIIatbp1384 and SauIIIA atbp1433) aswellasBamHI restriction sites provided by random insertions ofanQtfragment (26) into the repA gene(18). DNA segmentsstartingattheSpeI site(bp 680; Fig. 3) and containingthe repA promoter andvarious portionsof thegene wereclonedintopUC19,in the direction opposite that of the lac promoter present on this plasmid.

Stop codons in all threereadingframeswereintroducedatthe 3' end ofsomeof the truncated genesby insertion ofthe Q fragment.Thefragmentswerecalled D1(endingatbp1028), D2 (bp 1188), D3Q (bp 1335), D3TQ(bp 1384), D3BfQ (bp 1433),D3QfQ(bp 1458),D4Qt(bp1488), DSM (bp1602), D6Q (bp1692), andD7(bp 1705). Thecorresponding plasmidsare calledpUC19-D1, pUC19-D2, pUC19-D3TQ, etc. Theplas- mids were finally introduced by transformation into C600 cells.

PlasmidpUC19-D7-D3BQ1wasconstructedbycloningthe Pvu IIfragment ofD3Bf, containingatruncatedrepAgene followedby theft fragment,into the HincII site ofD7.The resultingplasmid pUCD7-D3BfQ carries both the wild-type and the truncated repAgene, each of themexpressedunder

*Presentaddress: Institut furAllgemeineMikrobiologie, BernUni- versitat, BaltzerStrasse4, 3012Bern, Switzerland

8923 Thepublicationcostsof this articleweredefrayedin partby pagecharge payment. This articlemusttherefore beherebymarked"advertisement"

in accordance with 18 U.S.C.§1734solelytoindicate thisfact.

Proc. Natl. Acad. Sci. USA 89 (1992) 4F

2(X)

luinchl par

DnaA

4X) 600 80(X0 I 0

inC reLpA

IHF RSI RS2 RS3, RS4

T A

IRI IRZ

lRNA\A

FIG. 1. Essentialfeaturesof thepSC101 originregion. The scale is in base pairs.

thecontrolof a repA promoter. Thisplasmidwasintroduced intothe recA HB101 strain.

Wecloned intopUC19 orpBR322 synthetic DNAoligo- nucleotides, flanked by restriction sites,that wereidentical with RS1 plus RS2 (pUC-AG), RS3 (pUC-AG3), RS4/IR1 (pUC-SP), and IR2 (pBR-Pr). The DNA fragments usedin thisworkastargetsites for thebinding of RepAwere cutout oftheseplasmids by usingBamHI-EcoRIorBamHI-HindIII (Fig. 2A).

RESULTS

Effects of C-Terminal DeletionsontheBiding ofRepA. We tested the ability of pSC101 RepA proteins with various C-terminal deletionstobindtotarget DNAsites.ApSC101 fragment containing therepA gene under the control of its own promoter had already been cloned in the high copy number vector pUC19 (plasmid pMX3; ref. 27). The trun- cated repAgeneswereclonedsimilarly (Fig.3 andMaterials andMethods). Crudeextractsofproteins from cells harbor-

A RS1+RS2:

aattcgagctcggtacccggggatcCAAAMGTCTAGCGGAATTTACAGABGT

qctcqaqccatgqqcccctaqGTTTCCATCGCCTTAAATGTCTCCCAGATCGTCTTAAATGTtcqa

ingsuchplasmids providedenough RepAproteintomonitor its binding to the repeated sequences by gel retardation experiments.

TodetectDNA-binding activity, crude extractscontaining intact or truncatedRepA were incubated with targetDNA fragments. The DNA targets used were either afragment containing the RS3 repeat sequence, afragment containing both RS1and RS2, afragmentnamedRS4/IR1, containing the sequencesbetween RS3and IR2, ortheinvertedrepeat sequence IR2.Thesefragments (described in Materialsand MethodsandFig. 2A) were labeledwith32P by filling inthe ends with the Klenow fragment ofDNA polymerase. The protein-DNA mixtureswereloaded onnondenaturing poly- acrylamidegels. A summaryoftheresultsisshowninFig.3.

Binding ofRepAto theinversely repeatedsequencesIR2 andIRL.Fig.4Ashows theautoradiogram ofaretardationgel monitoringtheability ofthetruncatedRepAproteinstobind tothe secondinversely repeatedsequenceIR2and decrease its mobility in the gel. The fragment was retardedby cell extractscontainingtheentireprotein (D7; lane 10),aswellas byextractscontainingtruncatedproteinswithdeletions ofup to 89 amino acids (lanes 5-9). The protein from plasmid D3TH, which lacks the last 105 amino acids (lane 4), and those withlonger deletionsdidnotbindtothe targetsite.In

RS3:aattcgagctcggtacccggggatcCAAAG6TCTAGCAAATMTACAGA gctcgagccatgggcccctagBMTTCCAGATCGTCTTAAATGTCTtcga RS4/IR1:

gatcCCACAACT 6AAGACTMTATTATCA1TTACTAGCCg BGTBTTBAGMCCTTTTCCTGATCATTAATASTAACTGATCG6ctta IR2:gatcCATCTCAATTGGTATASTGATTAAAATCACCTAaACCAATTGAGAT~a

GTAGAGTTAACCATATCACTAMTTTTMThSTCTSGTTAACTCTACttcga

B

5' -.3' 5'-.3' 5S^..3' 5'-.3' 3'-45'

59 ^- 39 39*--59

C AAA GGTCTAS CGGAATTTACAGA

GGTCTA6CAGAATTTACA

C AAA BGTCTAB CAGAATTTACAGA AAAAGGACTAGTAATTAT

AGATGGGCTAGTCAATG

AATTGGTATAB TGATTAA

AATTGBTCTAB GTGATT

FIG.2. (A) Sequences of the RepA bindingtargetsitesused in band-shift experiments. Bold ^uppercase letters represent pSC101

sequences; lowercase letters represent foreign DNA sequences.

Starsindicatethestronglyhomologous regions shown in Fig. 2B. (B) Directly repeated- and inversely repeatedsequences. Fortheright

armsof theinverserepeats,IR1-R andIR2-R, the^sequenceshown isthereversecomplement of the originalsequence.Only the central part of the IR2 palindrome is shown. Boxes indicate strongly homologous regions.

Dl I

D2 D3Q D3m D3BQ D3Q(- D40 D5Q

AA Bandshift missing IR RS

224 - -

170 - ^-

121 - -

105 ^{- -}

89 + +

80 + +

70 + +

32 + +

21 + +

- + +

D7

repA

Preps

680 1700

FIG. 3. Map ofthe repAgeneregion, with itspromoter,and ofthe variousdeletionfragments used in thispaper.The number of amino acids deleted in each fragment is shownaswell as the resultsof band-shiftexperiments involving either thedirectly (RS)orinversely (IR) repeatedsequences.A" + "indicates thataretardedbandwas

observed. No attempt wasmade at quantitating the results. AA, amino acids.

2(y

RS1RS2 RS3IR1-L IR1-R BR2-RAR2L

8924

Microbiology:

4 5 6 7 8 9 10

FIG. 4. Band-shift experiments with thevarious deletion pro- teins. Crude extracts from cellscontaining the indicated plasmid and producingthe different proteinsindicatedinFig. 2 were incubated with IR2 (A) or with RS3 (B) before they were loaded on the same 7.5% polyacrylamide gel. Lanes: 1, no plasmid; 2, pUC19; 3, pUCD3fl(121aminoacidsdeleted); 4,pUCD3Tfl(105 aminoacids deleted); 5, pUCD3BQ (89 amino acids deleted); 6,pUCD3Qfl(80 amino acids deleted); 7, pUCD4fQ (70 amino acids deleted); 8, pUCDSQ (32 amino acids deleted); 9, pUCD6fl (21 amino acids deleted); 10, pUCD7 (no deletion).

the retarded bands, the mobility of the DNA-RepA com- plexes increased as the protein fragments became shorter.

The segment that we callhereRS4/IR1 is composed of the 19-bp RS4 sequence, which is partially homologous to RS, and the 26-bp IR1 palindrome, which overlaps RS4 and extends 15 additional bp to the left of RS4 (Fig. 2A). This regionbinds RepA in vitro (Fig. 5C, lane 4) and is partially protected by RepA in footprinting experiments (12, 14). The results were similar to those obtained with IR2 (data partially shown;Fig. SC, lanes 3 and 4): the last 89 amino acidswere notrequiredfor binding.

Binding of RepA to thedirectly repeatedsequencesRS3 andRSJ plusRS2. Resultsshowing thebindingof the RepA fragments to thedirectly repeated sequences RS3 (Fig. 1) are shown inFig. 4B. They are similar to those obtained with IR2:

thelast89amino acids are not requiredfor binding.

Complexes obtainedwith theD5Q extract(proteinlacking 32 amino acids) and the D6W extract (protein lacking 21 aminoacids) did, however, show a double band whose upper component had a mobility that was slightly less than that obtained with the entire RepA (Fig. 4B, lanes 8 and 9). This resulthas notbeen explained. The deletionsmight, in those cases, have affected thefoldingof theprotein or itsbinding properties,factorsthatintervenein themobilityofa DNA- protein complex. This phenomenon was not observed with the IR2probe (Fig. 4A).

Similar results were obtained when the DNA segment containing RS1 plus RS2 was used as a probe. The RS2 sequence does not,byitself, bindRepAand,attheconcen- trations used in ourexperiments, probably does not bind it

B ^C

_ IAW

1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6

FIG. 5. Crudeproteinextractswereincubated with IR2(A), RS3 (B),orRS4/IR1 (C). Lanes: 1,noplasmid; 2, pUC19; 3,pUCD3BQ (89 aminoacidsdeleted); 4, pUCD7 (wild-type protein);5,pUCD7- D3BQ (wild-type and deleted protein); 6, mixture of pUCD7and pUCD3Bfl.

when associated with RS1-thatis tosaythat the mobility behaviorof thecomplex indicatedthatonlyoneproteinwas

boundtothefragment (data^not shown).

The DNA targetscontaining RS3orIR2 haveroughlythe samesize. Yettheirmobilityafterbindingisquitedifferent;

the IR2complexis much slower than the RS3complexwhen runonthesamegelunderidenticalconditions,whereasthe

fragments

4A andB). It seems likelythat this difference is due to a difference in the sizeorin the amount ofprotein bound to each target(28).

Fig. 2B shows the sequence homologies between the differentbindingsites. A strong7-bp homology(GGTCTAG) emergesbetween them. This suggests that this shortconsen- sus sequence might be the primary binding site for the protein.

DoesRepA Bindas aMonomer or as a Dimer? Insolution thepurifiedRepAproteinismostlyindimeric form (12). We soughttodetermine whetherRepAbindsas amonomer oras a dimertoits different specific sites. The shortenedRepA derivative encodedby D3Bf still bindstoall thesitesand,by virtue of its lower molecular mass, its DNA-protein com- plexes migrate faster in a gel than those made with the wild-type protein (Fig. 5, lanes 3 and 4). We extracted total proteinsfrom HB101 cellsharboringtheD7-D3BUplasmid.

This plasmid (see Materials and Methods) produces two proteins: the intact RepA and the truncated one, both under the control ofarepA promoter. After incubation of the crude extractwiththe inverted sequence IR2,threecomplexes of differentelectromobilities were seen on agel (Fig. 5A, lane 5).Two ofthem correspondtothecomplexes formed with the full-length RepA and with the truncated RepA alone. The third band has an intermediate mobility. We interpret this resultasindicating that the firsttwobandsareproducedby homodimers of either intactRepAortruncatedRepAbinding tothe target DNA and that the third band results from the formation of heterodimers between the two forms of the protein. When two crude extracts containing either the wild-type RepA alone or the truncated RepA alone are mixed, the intermediate band does not appear. Fig. 5Cshows similar results, using RS4/IR1as the target site.

The results are quite different when thedirectlyrepeated sequences RS3 or RS1plus RS2 are used as a target: noband of intermediate mobility was detected on the gels. Fig. SB shows the results withRS3 as the target; similar resultswere obtained with RS1 plus RS2 (data not shown). No difference wasnotedwhen, instead ofthe two proteinsoriginating in the samecell, crudeextractscontaining each of the twoproteins alone weremixed.

As observedpreviously, complexes made with IR2(Fig.

5A, lane 4)migrate much moreslowly than those made with RS3(Fig. SB, lane4).

These experiments also indicate that heterodimers are formed when the protein subunits are synthesized in thesame cell but that they cannot be assembled, in appreciable amounts, underourconditions,afterextraction. The failure of heterodimers to be formed and bind to the IR sequences when two extracts each containing only one form of the protein are mixed shows that dimerization must precede binding. The results also show that the dimerization domain of theprotein doesnotinclude thelast C-terminal 89 amino acids.

DISCUSSION

The data presented here show that 89 amino acids at the C-terminal end of the RepA protein are not needed for bindingin vitro toeither thedirectly repeatedsequences or theinversely repeatedsequences in theoriginofreplication ofplasmid pSC101.Datanotpresentedhere have shown that

A B

_.l

_ mo -w s

2 3 4 5 6 7 8 9 10 12 3

A

44,k-:- AM

Proc. Natl. Acad. Sci. USA 89(1992) only very short deletions (2-12 amino acids) can be tolerated

atthe N-terminal end.

In vivo replication of the plasmid has more stringent re- quirements: none of the plasmids with fi insertions, from which the truncated proteins shown in Fig. 3 were derived, were able to replicate (18). Amutation in the third codon ofthe genealleviates therequirementfor theIHFfunction inrepli- cation (29). A point mutation in codon 56 produced the temperature-sensitivemutantpHS1,whereaspointmutations in codons 92, 93, and 96 resulted in suppression of that mutation and inahighcopynumber (9, 27).Thus, the entire protein, or nearly the entire protein, is needed for in vivo replication.

Inthe initial constructions, the truncatedproteins carried variable tails of foreign origin, due to adventitiousextensions of the open reading frame caused by the construction. We observed that such tails often resulted in erratic binding behavioroftheprotein. Theintroduction ofanfi fragment, with stop codons in all threereading frames,atthe end ofeach truncated sequencerestored coherentbinding properties.

Wefoundthat, with either one of theinversely repeated sequences as aprobe, extracts from cells producingboth a wild-type RepA protein and a truncated one yieldedthree bandsinabandshift assay:twowith mobilitiesequaltothose produced by extracts inwhich eitherone of theproteinsis presentalone and one of intermediatemobility.Weinterpret this intermediatebandasresultingfrom heterodimers formed between the^twoproteins.Whendirectly repeatedsequences were used as a probe, only two bands, with the mobilities expectedfor each of thetwoproteins provided alone, were seen.Ourinterpretationisreinforcedbyconsideration of the mobility ofprobes RS3 and IR2 after incubation with an extract containing wild-type RepA. The two probes have nearlythesamelength(Fig. 2A)and thesamemobility (Fig.

4); yet, the protein-bound IR2migrated much more slowly than the protein-bound RS3. This is consistent with IR2 complexingwithadimer and RS3 withamonomerofRepA.

A similar migration behavior for these two probes was observed whenpurified RepAproteinwas used(14).

Churchward'sgroup has observed that thepurified pSC101 RepAproteinwaspresentintwoforms with differentbinding properties (G. Churchward, personal communication). They havefound that therewasnocompetitionfor thebindingof thepurified proteinbetweenaDNAfragment containingIR2 andonecontainingthe RS1direct repeat,althoughbothwere bindingsomeprotein. This suggests that thepurified protein contains two populations, each binding to onetarget only.

Wehaveconfirmed theseobservationswithpurifiedRepA, usingthe segmentsshown in Fig.2A(14). Sugiuraetal.(12) have found that the purified RepA proteinis presentmostly indimeric form. Since^wefind that thedimeric form is stable and is not assembled efficiently in vitro, we conclude that both thedimericand themonomericformscoexistinthe cell and inpurified protein preparations.

Togetherthese dataindicate thatRepA bindstorepeated sequencesas amonomerandtoinversely repeatedsequences as adimer.They indicate further that dimers binddirectlyto inversely repeated sequences as opposed to a binding of monomersfollowedby dimerization. Our data also indicate thatapproximately one-third oftheprotein at the C-terminal end and afew amino acids at the N-terminal end are dis- pensablefor dimerization oftheRepA protein.

The method usedhere to demonstrate the occurrence of heterodimers had been used previously to determine the heteromeric subunit structure of proteins like GCN4, a yeast transcriptionfactor (30).

Wickner etal. (31) have obtained results indicating that the RepA protein of prophage P1 can bind to the repeated sequences of that

plasmid only

first DnaJ and then, in anATP-dependent reaction, DnaK.

They find that the monomer form is stable in dilute solutions.

Therearesomedifferences between the pSC101 and the P1 situations: in P1 the dimer form does not seem to have an active role and may be involved only in regulating the amount ofactive, monomeric form present in the cell. In contrast, in pSC101, dimers and monomers seem to have different func- tions:autoregulation of repA for the dimer and activation of replication for the monomer. Adding ATP did not improve thebinding reaction (unpublished results). We find that RepA produced in dnaK mutant cells binds poorly to directly repeated sequences but binds normally toinverselyrepeated sequences(F. Keppel, personal communication), suggesting, asin the caseofP1,arolefor DnaK in the dimer-to-monomer transition, butwedonotfind any effectof dnaJ mutations.

Wehave found that asyntheticDNAfragmentcontaining onlythe entireIR1 siteor onecontainingthe RS4site,asthey weredefinedbySugiuraetal. (12), do notbythemselves bind RepA (data not shown). The RS4/IR1 fragment, which we have used as a probe, and which binds RepA efficiently, extends 19bptothe left of IR1and 15 to the leftofRS4. The footprintdata ofSugiuraetal.(12)andour own(14)indicate thatprotection againstDNase Idigestion conferred by RepA extends 9 bp to the left of IRL. It must, therefore, be considered that these 9bparepartof theRepAdimerbinding site. Ourbindingdataas wellasthe competitiondatamen- tioned above indicate that RS4 isnot aproperbindingsite for the monomerofRepAand does notseem tohaveafunction of itsown.

The resultspresented hereindicatingthatdimers andmono- mersof theproteinareformed in thecell,arestableinvitro, and have differentbinding properties, as wellas the data of Wickneretal. (31), mightthrowa newlightonsomeprevi- ously acceptednotions. Thus,itseems nowwidelyaccepted thatincompatibility,forplasmids oftheP1-pSC101type, isnot duetosequestration oftheprotein(32, 33). Thefact, however, thatinitiation ofreplication is due not to the entire poolof proteinbutto aminorform,theregulation ofwhichispoorly understood, could call this belief intoquestion. Various au- thors(12-14)havedemonstratedastrongerbinding ofpSC101 RepA to inversely repeated sequences than to directly re- peated sequences. We, now, might wonderwhether this is entirely due to a higher affinity of the protein for these sequencesor tothebalance ofmonomerand dimerformsin theextract. Insupportof theoriginalinterpretation,wehave found that the protein was more stably bound to inversely repeatedsequences thantodirectly repeated sequences (14).

Wewould liketothankGordon Churchward for communication of unpublished results;ThomasGoebel,FranceKeppel,XiaGuixian, HenryKrisch, and Pierre Prentki forhelpful discussions; Maryse PougeonandDanyRifat for technicalassistance;and EdouardBoy de la Tourand Otto Jenni fordrawingsandphotographs.This work wassupported byGrant 31.25698.88from the Swiss National Science Foundation.

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