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Cell, Vol. 52, 743-755, March 11, 1988, Copyright 0 1988 by Cell Press

Duplex Opening by dnaA Protein at Novel Sequencesin Initiation of Replication at the Originof the E. coli ChromosomeDavid Bramhill and Arthur KornbergDepartment of BiochemistryStanford Universi ty School of MedicineStanford, California 94305

SummaryThree tandem repeats of a 13-mer in the AT-rich regionare essen tial to the unique replication origin of E. coliand of remotely related Enterobacter iaceae. Theseiterated seq uenc es are identified by deletion analys isand sensit iv i t ies to endonucleases as the si te for ini-tial duplex opening by the initiator dnaA protein. Thisopen com plex requires ATP and 38% for optimumformation and stabi l i ty. The subsequent dnaCdepen-dent entry of dnaB hel icase to form a preprimingcomplex stabi l izes the open structure, blocks cleav-ages by a restr ict ion endonuclease in the 13-mer re-gion, and broadens the endonuclease cutt ing pattern.We propose that dnaA protein recognizes and succes-sively opens the 13-mer sequences, thereby guidingthe entry of dnaB hel icase into the duplex preparatoryto priming of replication.IntroductionIn Escher ichia col i , a unique sequence, or iC, serves as thesite for the initiation of chrom osom al replication duringnormal growth. The functional elements of or iC are con-tained within a 245 bp sequence highly conserved amongthe Enterobacter iaceae (Oka et al ., 1980; Zyskind et al .,1983). Four 9 bp sequ ences (dnaA boxes ) bind the initia-tor dnaA protein (Ful ler et al ., 1984). Howev er, the functionof a 50 bp region at the left end of oriC, also strikinglyconserved, has not been identif ied.

Using puri f ied enzym es to repl icate supercoi led or iC-containing templates, the sequence of events precedingDNA synthesis has been divided into four stages: f i rst,recognition of oriC by dnaA protein a nd coope rative bind-ing to the four dnaA boxes; secon d, incorporation of dnaBand dnaC proteins into a prepr iming complex; third, bi-directional unwinding by the dnaB he licase to generatetwo forks; and fourth, pr iming by dnaG pr imase for chainelongation by DNA polymerase I l l holoenzyme (Kaguniand Kornberg, 1984; Ogawa et al ., 1985; van der Ende etal ., 1985; Baker et al ., 1986, 1987; Funnel l et al ., 1987).Protein HU stimulates the ear ly stages (Ogawa et al .,1985), and single-strand binding protein (SSB) and gyrasemust be present dur ing opening of the duplex to stabi l izethe single-stranded DNA and rel ieve the torsional stress(van der Ende et al ., 1985; Baker et al ., 1986, 1987). Nega-tively supercoiled DNA has a greater aff ini ty for dnaA pro-tein (Fuller and Kornberg, 1983) and is required for oriC Present address: Department of Bio logical Sciences, StanfordUniversity, Stanford, Californ ia 94305.

templates to be repl icated (Funnel l et al ., 1986). In vi tro,a sharp increase in the rate of prepr iming complex forma-tion is observed at around 30% as the temperature is in-creased (van der Ende et al ., 1985).

No direct evidence has previously been obtained to indi-cate that any part of the template becomes single-stranded at very ear ly stages pr ior to dnaB hel icase ac-t ion. Here we identi fy a 13 bp sequence present as threerepeats in or iC, and demonstrate that these sequencesare opened in the prepr iming complex. With the single-strand -specific nuclease Pl from Penicill ium citrinum(Kowalski , 1964; Camilloni et al ., 1986) as a probe, w eshow that the ini t ial duplex opening occurs within theserepeats, and is catalyzed by the dnaA ini t iator protein.We also report the construction of deletion der ivativesof or iC and their activi ty in each step of ini t iat ion. Theresults strongly support the proposal that dnaA proteinrecognizes the 13-mer sequences and, fur thermore, sug-gest a mechanism of successive opening of al l three 13-mers preparatory to dnaB entry.ResultsThree 13 Base Tandem Repeats in or iCThe boundary at the r ight end of or iC (Oka e t al ., 1980)coincides with the end of the r ightmost of four 9 bp se-quences (boxes) bound by dnaA protein (Figure 1). Theleftmost box is about 60 bp from the left boundary of or iC.Thus, the absolute requirement for the dnaA protein in ini-tiation in vivo and in vitro (Fuller and Kornbe rg, 1983) canaccount for the role of the r ighthand segment of or iC, butnot of the leftmost 60 bases. Comparison of the or iC se-quences of the Enterobacter iaceae (Zyskind et al ., 1983)reveals that this leftmost sequence is highly con servedand contains three tande m repeats of a 13 bp moti f (Table1); the consensus sequence indicates that 11 of the 13 po-si t ions are highly conserved. Sequences f i t t ing this con-sensus also occur as two tandem repeats at a distance of7 bp from the dnaA box within the or igin of plasmidpSC 101; as with oriC, initiation at the pSClO1 origin re-quires dnaA protein (Hasunuma and Sekiguchi, 1977;Frey et al ., 1979; Felton and Wright, 1979). Computer-aided search of sequence data bases of E. col i and i tsphages and plasmids revealed no other tandem repeatsof this 13 base consensus other than those in Table 1 . Ofthe few single close matche s identi fied, two are notewor-thy. One occu rs in the dnaA gene promoter (Hansen et al .,1982) only 17 bp from a dnaA box (Ful ler et al ., 1984); theother is near the 16 kd gene promoter, 10 bp from i ts dnaAbox (Buhk and Me sser, 1983).

Consistent with the consensus deduced from the E. col isequences, the few di fferences between the left ends ofEnterobacter iaceae OriC sequences occur almost exclu-sively at ei ther of the two nonspecif ic posi t ions in the se-quence. Deletion of ei ther the left 13-mer or part of themiddle one inactivates or iC; a C-T change at the fourthbase of the middle 13-mer reduces or icfunction. No effect

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Ce l l74 4

minimal orig ln~13 merspsc101ori

16 kdgene

Figure 1. Locations of the 13-mer R epeatsThe diagram indicates 13-mers ( left [L], middle [M]. r ight [R] by largesolid arrows, 9-mer dnaA boxes by stippled boxes, RN A polymerasepromoters by lightly stippled arrows, and pSClO1 rep prote in b indingsequences by small arrows. The figure is not drawn to scale.

is observed when a 4 base insertion is made between theleft and middle 13-mers (Oka et al ., 1980).Probings with EndonucleasesOpening of the Duplex Is Catalyzed by dnaA ProteinFunnel l et al . (1987) observed that the 13-mer region oforiC is involved in the prepriming com plex but not in anear ly or iC-dnaA complex. The sharp transi t ion in temper-ature profile (van der Ende et al., 1985) and the require-ment for a negatively supercoi led template for prepr imingcomplex formation (Funnel l et al ., 1986) both suggest thatthe ini tial opening of the duplex occurs at an ear ly stage.The AT-r ich nature of the 13-mers suggests a possible rolein melt ing the duplex DNA . To test this directly, Pl en-donuclease was used to probe ini t iat ion intermediates.This nuclease, simi lar to Sl in i ts speci f ic i ty for cleavageof single strands (Kowalski , 1984) has the advantage ofacting at pH 7.6, the optimum for or iC repl ication.

Action by dnaA protein sensi t izes or iC-containing su-percoils in the 19mer region to linearization by Pl en-donuclease. Two types of Pl-sensi t ive complexes can be

OI 0 50 100 150 200 250 300 350dnaA PROTEIN (ng)

Figure 2. Pl Nuclease Linearization of an oriC Plasmid Depends ondnaA Prote inThe reactions, contain ing 150 fmol of pCM959 supercoils in 50 u l wereas described in Experimental Procedures. Open complex formationand Pl nuclease probing were at 38%.

distinguished: an open complex, which requires onlydnaA protein for i ts formation and a high temperature (e.g.,30%) for both i ts formation and i ts stabi l i ty; and theprepriming com plex, which requires dnaB and dnaC pro-teins in addit ion to dnaA protein for formation, but which,once formed at a high temperature, is stable to chi ll ing to16% or even to 0C.

Several character ist ics of the open complex indicatethat it is a true intermediate in initiation: first, the need forhigh temperature; second, the requirement for dnaA pro-tein levels of 20-40 monome rs per ofiC sequence (Figure2), similar to those in the overall reaction (Fuller and K orn-berg, 1983); third, the extent of Pl sensi t iv i ty (up to 50%of the input DNA), suggesting that i t is not a minor by-product; fourth, the requirement for the or iC sequence(see below); and f i f th, the inactivi ty of the ADP form ofdnaA protein in open complex formation, despite the addi-t ion of ATP to 5 mM (Sekimizu et al ., 1987; and see below).Inclusion of H U prote in greatly reduces the backgroundlevel of nonspecif ic cutt ing by Pl, but its known stimula-

Table 1. Thirteen Base Pair Repeats and Deduced Consensus SequenceDistance from dnaA

Si te Location Sequence (5 to 31 Match Box (WoK a L GATCTATTTATTT 10111

M GATCTGTTCTATT 10111R GATCTCTTATTAG 9111 13

oripSClOl~ L GATCTATTCTTTT 11111 7R TATCTTTTTTTAT 9/l 1

dnaA promoterC GATCTTCTGTTTC 9111 1716 kd gene promote? GATCGGCTTTTTT 9/l 1 10Consensus GATCTnTTnTTTTThe locations are indicated in Figure 1. The orientation of each repeat is such that the arrowheads in the figure correspond to the 3 end of thesequence given here. The predom inant or only base occurring at a given positio n is indicate d in the consensus; where no particular basepredom inates, n is used, The number of matches to the 11 specific positions in the 13-mer consensus is indicate d, as is the distance from thenearest d naA box.a Meijer et a l., 1979; Oka et a l., 1980.b Vocke and Bastia, 1983; Churchward et a l., 1983; Armstrong et a l., 1984; Yamaguchi and Yamaguchi, 1984.c Hansen et a l.. 1982.

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dnaA-Catalyzed Duplex Opening74 5

Haa l l l Ava l-I I

(A)(c) &qGoG q?G-jGq~GoG& U N Hind l l lr

dnaA --@@--@@ oc WCu 9-mer dnaA - @ @ -

1 pCM959 )W SnaBl EcoRV--

& q9 & ,j9

- LINEAR\ 13.mers

m - 13.mers

13.mars a

IL- TR

Figure 3. Mapping Pl Cleavage Sites to the 13-mer Region of OXOpen complexes (OCs) or preprim ing complexes (PPCs) were formed as described in Experi menta l Procedures in B-fold reactions, each containi ng750 fmol of pCM959. Contro ls lacking dnaA prote in were a lso probed with Pi nuclease. DNAs were dephosphorylated and end-labeled using T4polynucl eotide kinase before restriction analysis. (A) Location of the relevant restriction enzyme sites in pCM 959 DNA near oriC. (B) Native gel show-ing that the predominant cuts l ie near the left end of ofiC. Samples of Pl- l inearized. 5 end-labeled DNAs were restr icted with e ither SnaB l or EcoRV,electrophoresed in a 1.2% agarose gel, and autoradiographed. The posit ions of the Bglll(13-mer) s ites in oriC are indicated for each case, determinedby comparison with double d igests (not shown). (C) Mapping cleavage sites on the lower strand with respect to the Aval and Haelll s ites. Samplesof PI-l inearized, 5 end-labeled DNAs were restr icted with e ither Haelll or Aval, e lectrophoresed in an 8% polyacrylamide denaturing gel, and au-toradiogra phed. The positions of the 18mers relative to the Aval site are indicate d by the solid arrows (oriented as in Figure 1). The Aval fragmentsare 1 base longer than the Haelll fragments. (D) Mapping Pl c leavage sites in the upper strand. Samples of PI- l inearized, 5 end-labeled DNAswere restr icted with Hindll l and electrophoresed on a 6% polyacrylamide denaturing gel. The solid arrows (oriented as in Figure 1) indicate thelocations of the Id-mers. The lane of the sample of open complex plus dnaA was exposed for only 1 day; the other lanes w ere exposed for 4 days.

tory effects on ini t iat ion (Dixon and Kornberg, 1983) arediff icul t to measure by Pl sensi t iv i ty.The Sites Cut by PI N&ease Lie in the Id-mersTo determine the location of the sites cut by Pl, the uniquerestr iction targets for SnaBl and EcoRV in pCM959 wereused as reference points (Figure 3A). Fol lowing dephos-phorylation, the 5 ends gene rated by Pl linearizationwere labeled with 32P by T4 polynucleotide kinase. DNA swere then restr icted with ei ther SnaBl or EcoRV , electro-phoresed in a 1.2% agarose gel, and autoradiographed(Figure 38). The great major i ty of the si tes cut by Pl areclustered together at the left end of or iC, near the Bgl l ls i tes. No signi f icant l inearization was detected at anyother point in or near oriC. The full-length linear mole culespresumably result from labeling at nicks introduced by Plnuclease, since they are observed even fol lowing over-digestion with the appropr iate restr iction nuclease. Theyare mos t l ikely distributed throughout the templates.

More precise mapping of the predominant si tes was ob-

tained by using Aval, Haelll, and Hindlll restriction siteslocated close to the 13-mers in or iC (Figure 3A) and analy-sis on denatur ing polyacrylamide gels. The locations o fthe major si tes of Pl cleavage are al l wi thin the 19mer se-quences (Figures 3C and 3D). The cut si tes in the opencomplex are predominantly clustered in the middle 13-meron the upper strand, yet widely distr ibuted in each13-mer on the lower strand. This may reflect a protectionof the upper strand by dnaA protein.dnaA Protein A/one Confers PI Sensit iv i ty tothe It-mersThat the specif ic i ty for the AT-r ich 13-mer regions shoulddepend on recognition by dnaA protein is suggested bytheir proximity to a dnaA box and also by the absence of13-me rs from origins th at require proteins other than dnaA(Bramhill and Kornberg, subm itted). The possib ility that acontaminant activi ty in the dnaA protein preparation mightcontr ibute to recognition of the 13-mer sequences hasbeen removed by an improved pur i f ication procedure,

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Table 2. IHF Can Replace HU Prote in forOpen Complex Formation

dnaA -@@-HU @@--

Additions L inear Molecules (o/o)HU and dnaA 47HUa 9IHF and dnaA 52lHFa 10Reactions were as described in Experimental Procedures. Prote in ad-dit ions, where indicated, were: dnaA, 240 ng; HU, 67 ng; IHF, 60 ng.a When neither IHF nor HU is present, a h igh level (40%) of dnaA-indepe ndent lineari zation is observed. This background cutting is dis-tr ibuted around the p lasmid and is not localized to the 19mer region(see rightmost lane, Figure 4).

which yields dnaA protein containing no detectable impu-r i t ies (Sekimizu et al ., submitted); both the Pl-sensi tiz ingand repl ication activi t ies copur i fy on FPLC (Pharmacia)gel f i l tration and Mono-S ion exchange chromatography(data not shown).In addit ion, integration host factor of E. col i ( IHF) (Nashand Robertson, 1981) can replace the HU protein at simi-lar levels (Table 2) withou t altering the overall exte nt of thednaA-dependent reaction. These two simi lar , basic pro-teins are interchangeab le in reducing the nons pecific Plbackground cutt ing. The fact that they support the dnaA-dependent l inearization with simi lar effic iencies suggeststhat nei ther contr ibutes to the sequence specif ic i ty. Th edramatic effect of low levels of HU or IHF (sufficient to coatonly 3% to 5% of the template) in reducing the nonspecif iccutt ing reaction by Pl nuclease indicates the profoundconformational changes induced by low levels of ei therprotein. IHF also substi tutes for HU protein in the stimula-t ion of the reconsti tuted repl ication of or iC templates (D.Bramhi l l, T A. Baker, and A. Kornberg, unpubl ished data).

Final ly, the major cleavage si tes for Pl endonucleaseinduced by dnaA protein in the presence of HU are identi -cal to those induced by dnaA protein alone (Figure 4). Inthe latter case, the extent of 13-mer-specif ic cutt ing isonly one-fif th that observed in the presence of HU or IHF.In addit ion, in the absence of HU and IHF, minor dnaA pro-tein-independent Pl cleavage si tes are also detectableoutside the 13-mer region of 0% (not shown).Nucleotide Requirements for Opening of the DuplexThe dnaA protein has a very high aff ini ty (Ko = 30 nM)for ATP, ATPyS , and AD F, with which i t forms very stablecomplexes (Sekimizu et al ., 1987). Both the stabi l i ty ofdnaA protein and i ts activi ty are profoundly affected bynucleotide binding: the ATP and ATPyS form s are both ac-t ive in repl ication ( in the presence of 5 mM ATP), whereasthe ADP form is inactive and fai ls to induce a Pl-sensi t ivestructure in or iC (Sekimizu et al ., 1987).In addit ion to the requirement for micromolar levels ofATP open complex formation requires a level of ATP near5 mM for optimum eff iciency. Both dATP and CTP cansubsti tute for ATP in open complex formation (Table 3) butGTP UT P, dTTP, and dCTP fai led to support duplex open-ing (data not shown). The ATP analogs ATP$S, AMPP NP,and AMPPCP at 5 mM cannot replace ATP completely.

L

I13.merr M

I

R

1

Figure 4. Comparison of Pl Cleavage Sites in oriC Induced by dnaAProtein in the Presence or Absence of HU Prote inReactions contain ing 150 fmol of pCM959 DNA were incubated at36OC for 2 min with 67 ng of HU prote in or 240 ng of dnaA prote in orboth or neither, as indicated. L inearization with 1.2 U of Pl nucleasefor 5 set was at 38% Following phenol extraction, samples were 5 end-labeled and restricted with Hindll l. Electrophoresis was on a 8%polyacrylamide denaturing gel. The locations and orientation of the19mers are indicated.

Table 3. Nucleotide Requirements for Formation of OpenComplex and Prepriming Complex

PreprimingOpen Complex (%) Complex (o/o)Full Minus Full Minus

Nucleotide Reaction dnaA Reaction dnaCATP 53 6 85 10CTP 50 7 5 6dATP 46 10 4 5None 8 10 5 5The dna A prote in was first incubated with 1 uM ATP for 15 min at 0%to generate the ATP form of dnaA prote in. Complexes were formed at38% as described in Experimental Procedures, with the indicatednucleotide present at 5 mM. Open complexes were probed with Plnuclease for 5 set at 38%; prepriming complexes were chil led to 16%and then probed with P l nuclease for 5 sec. The fraction of moleculeslinearize d by Pl was determ ined and is expressed as a percentage.

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$A-Catalyzed Duplex Opening

% 408;CL 35Pd 302z 25g 20Lx 15ig IO0

5a

plus 5 mM AMP-PCP

\-.- ATP aone

.-.I I I I I I1 2 3 4 5 6

dnaA-DEPE NDENT LINEARS ICATPI (percent-PM. )Figure 5. Eadie-Hofstee Plot of Dependence on ATP of Formation ofthe Open ComplexReactions were as described in Experimental Procedures. The ATPconcentration was varied from 0 to 5 mM; in one titration AMP PCP wasincluded at 5 mM. The proportion of molecules linearized by PInuclease was determined, and the dnaA prote in-independent back-ground (6%) was subtracted. Production of dnaA-dependent l inearmolecules was a measure of the in it ia l rate.

Howeve r, in the presence of these analogs, much lowerlevels of ATP can activate the reaction. Assuming that theexte nt of dnaA-depe ndent Pl l inearization is proportionalto the initial rate of duplex opening, more refined kineticanalysis is possible. This assumption is consistent withthe observed t ime course for the duplex opening reaction,which shows a 5 set lag fol lowed by a rapid approach tothe maximu m Pl sensi t iv i ty. In the presence of 5 mM ATPthe increase is more rapid ( t,/ , 2 4 set) than at lower ATPconcentrations (T,,, 4 8 set at 1 mM ATP) (data notshown). Analysis of these data by a Lineweaver-Burke (notshown) or Eadie-Hofstee plot (Figure 5) indicates two dis-t inct K, values for ATP: one high-aff ini ty interaction (K,< 1 PM) and one low-aff ini ty interaction (K, = 900 PM).The high-affini ty K, value cannot be estimated more pre-cisely because of the low level of the signal in this range.Simi lar plots and K, values are observed for CTP anddATP (data not shown). In the presence of AMPPCP at 5mM , a single apparent K, value for ATP is observed atabout 26 t.rM (Figure 5). This probably reflects saturationof the low-aff ini ty si te by AMPPC P where i t can substi-tute for ATP, combined with competi t ive inhibi t ion at thehigh-aff ini ty si te ; the V,, remains unchanged.Further Opening of the Duplex in thePrepr iming ComplexFol lowing incubation at a high temperature with dnaB anddnaC proteins, a prepr iming complex that also includesthese proteins at or iC can be isolated. Subsequent stagesof repl ication can then occur at 16%. Prepr iming com-plexes and possible subassem bl ies were probed with Pl

Table 4. Requirements for Formation of a PI-SensitivePrepriming ComplexComponent Omitted L inear Molecules (%)None 95dnaA IOdnaB adnaC 12HU 62None; no 36OC preincubationa aReactions, contain ing 150 fmol of pCM959 supercoils, were asdescribed in Experimental Procedures. Components were omitted asindicated. Probing with 1.2 U of PI nuclease was for 10 set at 16C.a The reaction was incubated at 16C, rather than 36OC, for 30 minbefore Pl probing.

nuclease at 16% (Table 4) to assess which compone ntsare required to stabi l ize the Pl-sensit ive open comp lex ata low temperature. Sensit iv i ty to Pl nuclease requireddnaA, dnaB , and dnaC proteins and an incubation atelevated temperature, and was stimulated by HU protein(Table 4). This agrees with previous studies of prepr imingcomplex formation using repl ication (van der Ende et al .,1985) or extensive unwinding (Baker et al ., 1986) as as-says . Along with the template becoming avai lable for pr im-ing and repl ication, this sensi t iv i ty to Pl nuclease sug-gests that part of the duplex DNA in the prepr imingcomplex has been me lted into single strands.

Mapping of the Pl cleavage si tes in the prepr iming com-plex was carr ied out as for the open com plex, and the si teswere compared with those in the open complex. The pat-tern of cuts is di fferent in the two types of complex, part ic-ular ly in the upper strand (Figures 3C and 3D ). Thegreater stabi l i ty of the prepr iming complex at the lowertemperature, as wel l as the di fferent pattern of cuts, im-pl ies DNA-protein interactions in the prepr iming complexdistinct from those in the open com plex.The nucleotide requirement for forming the stableprepr iming complex di ffered from that descr ibed for theini tial actions of dnaA protein. Whereas ATP dATF, andCTP are effective for formation of the open com plex, theprepr iming complex is uniquely dependent on ATP (Table3). Thus, at least one interaction at this step requires ATPin a manner dist inct from that observed in duplex openingby dnaA protein.The Prepr iming Complex Blocks the Bgl llNuclease Cleavages in or iCElectron microscopic measureme nts (Funnel l et al ., 1987)have shown the prepriming complex to be larger and lesssymme tr ical than the ini tial dnaA-or iC complex and tocover the D NA at the left end of or iC in the region of theBgl ll restr ict ion si tes. The plasmid pC M959 (Buhk andMesse r, 1983) contains four Bgl ll recognit ion si tes, two ofwhich l ie at the left end of or iC, overlapping the leftmostand middle 13-mers; the other two are 500 and 1800 bpdistan t from OX (Figure 6 ). Bglll nuclease can bind itsrecognition sequence at a low Mg*+ level (an optimumcondition for maintaining the prepriming com plex), but re-

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Ce l l74 0

B g l l lC 38 38 38 22

dnaA - 000dnaBdnaC

btc - -a+ CI..- --ML--.-. +I,

Figure 6. Prepriming Complex Formation Abolishes Cutting by BglllNuclease in oriCComplexes were formed at 36OC or 22C on 150 fmol of pCM959 DNAas described in Exper imenta l Procedures. Reactions were cooled to22OC, and 25 U of Bgll l was added and incubated for 5 min at 22oC.The magnesium concentration was then ra ised from 0.3 mM to 10 mMto allow cleavage for 5 set before being stopped with EDTA . Sampleswere extracted with phenol and end-labeled, using DNA polymeraseI large fragment, before electrophoresis through a 1.2% agarose gel.Fragments marked by arrows on the left can be generated only by cut-t ing in oriC; those marked on the right can be generated without cuttingin OK. Lowercase letters refer to the fragments indicated in the mapof pCM959 (above).

quires a high magnesium concentration (e.g., 10 mM) foreff ic ient cleavage.

Protein-DNA complexes were al lowed to form for 50min at a low Mg*+ level either at 38C or 22%, before ad-di t ion of Bgl ll nu clease. Incubation was continued at 22%at the low Mg*+ level to enable Bglll to bind (but no tcleave) i ts recognition sequences. Then Mg*+ was addedto 10 mM to ini tiate cleavage by Bglf l dur ing a 5 set per i-od. Ana lysis of the partial re striction produ cts (Figure 8)shows tha t signi f icant protection of the two Bgll l s i tes inor iC was observed only in the presence of HU, dnaA,dnaB, and dnaC p roteins and required incubation at anelevated temperature, the condit ion needed for formingthe prepr iming complex. Protection of the Bgll l s i tes inor iC after cooling at 22C is fur ther evidence of the sta-bility of the DNA -protein interactions in the preprimingcomplex.At 38% the open com plex does provide l imited protec-t ion (9fold) against Bgl ll c leavage (T A. Baker, D. Bram-

25

15 20 25 30 35 40TEMPERATURE (C)

Figure 7. Temperature Profiles of Formation of Open and PreprimingComplexes on Supercoiled pBSoriC+ DNA As Monitored by Pl Nucle-ase SensitivityOpen complexes were formed in 2 min at the temperatures indicatedand probed for 5 set wit h 1.2 U of PI nuclease. Value s for control reac-tions lacking dnaA prote in (240 ng) wereobtained at each temperature,and the difference was plotte d (open circles). P reprim ing complexeswere formed at the temperatures indicated for 20 min, then cooled to16C and probed with 1.2 U of PI nuclease for 5 sec. The dnaA pro-tein-ind epend ent values were subtracted, and the difference wasplotted (closed circles).

hi l l, and A. Kornberg, unpubl ished data). Two factors ma yexplain the low level of protection: First, the aff ini ty ofdnaA protein for the 19m ers may be simi lar to that of Bgl llfor i ts recognition sequence. Thus, Bgl l l might displace theequi l ibr ium, reducing open complex formation. S econd,al though numerous Pl cuts map in the leftmost 19mer,the major i ty l ie in the middle and r ightmost 13-mers. Sinceone 13-mer is suff ic ient for open complex formation (seebelow), then i t is possible that most open com plexes arestil l double-stranded in the leftm ost 13-mer and are there-fore Bglll-sensitive.Temp erature Profiles for Partial and FullOpening of the Duplex at 0r iCFormation of both the open and prepr iming complexes bydnaA protein requires an elevated te mpera ture (van derEnde et al ., 1985). Th e exten ts of open and prepr imingcomplex formation were measured at di fferent tempera-tures. To determine the minimum temperature for forma-tion of the open comp lex, probing with Pl nuclease wascarr ied out at the temperature at which the complex wasformed, inasmuch as these complexes are cold-sensit ive.dnaA protein was omitted from reactions at each tempera-ture to control for the influence of temperature on Pl activ-i ty. Open comp lex formation was clear ly evident at 23%(Figure 7). Prepr iming complexes were formed at a giventemperature, and then probed with Pl at 18%. Their ap-pearance required a temperature above 28%. This directmeasuremen t agrees well wi th observations using repli -cation (van der Ende et al ., 1985) or dnaB hel icase action(Baker et al ., 1986) as assays of prepr iming complex for-mation. Th us, open complex formation is observed at a

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;;;A-Catalyzed Duplex Opening

ORII/i,,,/ III ArcI PHENOTYPE+

Figure 8. Orig in Regions of Wild Type oriC and Mutant DerivativesArrows represent 15mers (see Figure 1). and stippled boxes represent9-mers. A4 bp insertion at the Hindll l s ite is indicated by a l ight stippledbox; deletio ns are indicat ed by gaps. Restriction sites used in the con-structions are indicate d (for details see Exper imenta l Procedures). Thefigure is not drawn to scale. The ori phenotype of each plasmid is indi-cated. pBSoriC+ DN A transformed a PO/A- host at least WOO-foldmore eff iciently than d id any of the oriC mutan t plasmids (no transfor-mants recovered). All p lasmids transformed a PO/A+ host with a s imi-lar efficiency (about 2 x lo4 per pg of DNA).

temperature at least 4% lower (at the transition midpoint)than that of the subsequent prepr iming complex forma-tion. The optimal Mg2+ level of 8 mM was used for form-ing the open complex, in contrast to the Mg*+ level of 0.3mM used for forming the prepr iming complex. Because ahigher Mg2+ level is expected to raise the temperature foropen complex formation, the temperature di fference (be-tween open and prepr iming complex formation), i f mea-sured at the same Mg2+ level , m ight be even greater than4% .

These f indings on the influence of temperature are inkeeping with the behavior of the mutated repl ication or i-gins (see below). On ly one 13-mer (theo retically, only asingle base pair) need be opened to generate a Pl-sensi t ive si te, and this can occur at 23%. Howeve r, to al-low dnaB to enter and generate a stable prepr iming com-plex, all 13-mers must be opened and kep t open. Sinceeach opened 13-mer el iminates more than one superheli -cal turn from the plasmid, successive openings are ener-getical ly less favorable-hence the requirement for ahigher temperature.Deletion AnalysesTo investigate the role of the 13-mers in more detai l , dele-t ions in or iC were constructed. The properties of each mu-tated or igin were then examined in vivo and in vi tro.In Vivo Function of or iC Requires Al lThree Id-mers and Four 9-mersEach of f ive mutations in the 0% sequence, deletion de-r ivatives of or iC ( lacking one, two, or al l three 13-mers, orthe r ightmost 9-mer), and an insertion between the twor ighthand 9-mers (Figure 8), completely destroyed the ca-paci ty of an oriC plasmid to transform a PO/A strain (datanot shown). The hos t, lacking pa/A function, cannot sup-port the pBR322-der ived vector or igin and must thereforedepend on or iC function to maintain the plasmid. The mu-

tant plasmids fai led to function in the PO/A- host, butcould transform the isogenic PO/A+ strain. These obser-vations are consistent with the or iC sequences extraordi-nary evolutionary conservation (Zyskind et al ., 1983) andits sensi t iv i ty to mutational changes (Oka et al ., 1980,1982, 1984 ). Seque ntial stage s o f initiation in vitro werethen examined to determine the basis for these mutationallosses of function.Formation of the Open Complex RequiresOne Intact Id-merThe mutated or iC plasmids were tested for their abi li ty toform open complexes with dnaA protein. Pl nuclease wasused at 38% to monitor com plex formation. Deletion ofthe left 13-mer (or iCA13L) or two 13-mers (ofiCA13LM)has no detectable effect ei ther on the level of induced Plsensitivity or on the amoun t of dnaA pro tein required (Figure9). Howeve r, deletion of al l three 13-mers (or iCAl3LMR)abol ishes duplex opening. Thus, the dnaA protein ap-pears to require at least one 13-me r to generate a single-stranded region; mere AT-r ichness, suppl ied by the con-tiguous DNA to the left of ofiC in ofiCAl3LMR, does notsuff ice for duplex opening. Mutations at the r ight end ofor iC have l i t t le effect on the reaction.Sites of P7 Sensit iv i ty in the Mutated or iCSequences Lie in Intact Id-mersMapping of the PI-sensi t ive si tes in the mutant or iginsconfirmed their location in the 13-mers that remained in-tact. The open com plex was formed with each D NA, l in-ear ized at 38% by Pl nuclease, and final ly analyzed in na-t ive gels and sequencing gels (Figure 10). In the native gel(not shown), vir tual ly al l (>95% as determined by densito-metric scanning) of the Pl cuts are seen in the 13-mer re-

0 60 120 160 240dnaA PROTEIN (ng)

Figure 9. Open Complex Formation on oriC Mutant DNAs, Monitoredby Pl Nuclease Sensitiv ityReactions were carried out as described in Experi mental Procedures,and contained 150 fmol of supercoiled DNA (see Figure 8) and dnaAprote in, as indicated, in 50 ~1. Following a 2 min incubation at 38OC,complexes were probed with 1.2 U of Pl nuclease for 5 set before be-ing quenched with 40 p l of 25 mM ED TA, 1% SDS. The proportion ofl inearized molecules was determined by densitometric scanning ofphotographic negatives of 0.7% agarose gels sta ined with eth id iumbromide. Values with oriCAl3LMR are identical to those with oriC com-plete ly deleted.

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DN A

.

LL

13.mers M

I *a *

R

I

Figure 10. Nuclease Cleavage Sites in Open Complexes Formed withthe oriC MutantsOpen complexes were formed with DNAs (150 fmol) and dnaA prote in(240 ng) as indicated and probed w ith Pl nuclease. L inearized DNAswere dephosphorylated and 5end-labeled using T4 polynucleotide k i-nase. Incubation with Hindll l generated fragments with in oriC of about200 bp (see Figure 8) that were resolved on a 8% polyacrylamide se-quencing gel (80 cm x 0.2 mm). The arrows indicate the posit ions ofthe 18mers in the wild type sequence, deduced from sequencing reac-tions and Bglll-Hind81 oriC fragments run in the same gel.

gion of or iC. The sequencing gel shows that in the caseof wi ld type or iC or oriCA13L, the cuts l ie predominantlyin the middle 13-mer. When 4 bp more ( the f i rst 4 bp of19mer M) are deleted from or iCA13L to give oriCA13LM,th e major si tes cut by Pl l ie in the only intact 19mer, ther ightmost, and not in the remaining 9 bases of the middle19mer. These results strongly support the idea that dnaAprotein speci fical ly recognizes the 13-mer sequence orsome portion of i t .Formation of the Prepr iming Complex RequiresA// Three l&mersMutational changes in or iC have a more profound effecton formation of the prepr iming complex than on formationof the open complex. Eff icient formation of the prepr imingcom plex requires all three 13-me rs (Figure 11). Even theloss of only one (or iCA13L) results in a very low level of

60 -

0 60 120 160 240dnaA PROTEIN (ng)

Figure 11. Prepriming Complex Formation with OK Mutant DNAs,Monitored by Pl Nuclease Sensitiv ityPrepriming complexes were formed at 38OC for 30 min with super-coiled DNA (150 fmol) as indicated. Reactions were chil led to 18OC andprobed for 5 set with 1.2 U of PI nuclease before being quenched with40 ul of 25 mM EDTA, 1% SDS. The proportion of l inear molecules wasdetermined from densitometric scanning of photographic negatives ofgels sta ined with eth id ium bromide. The dnaA-independent back-ground values (4%-10%) have been subtracted in each case. Value swith oriCA13LM are identical to those with oriCAl3LMR and with oriCcomplete ly deleted.

dnaA-dependent Pl sensi t iv i ty at low temperature. Theseresults are consistent with the temperature profi les ofopen and prepr iming complex formation (Figure 7). Nei-ther deletion of the rightmost 9-mer repeat (oriCA9R) no rinsertion of 4 bp between the two r ightmost 9-mers(ofiCV9R)appears to al ter the abi li ty to support formationof the prepriming com plex. Stil l, as noted earlier (Figure8) these m utants are inactive in vivo.Replication In Vitro Parallels PreprimingComplex FormationTwo reconsti tuted enzyme sy stem s were examined fortheir capacity to repl icate the mutant plasmids. In the solopr imase system (van der Ende et al ., 1985) a low level ofprotein HU is present (simi lar to the level of HU in the Plassays) to stimulate the reaction and RNA polymerase isabsent. In the other s ystem , higher levels of HU proteinpartially coat the plasmid and RNA polymerase transcr ip-t ion is required to activate the origin; RNAa se H is alsoneeded to maintain or iC specif ic i ty (Kaguni and Kornberg,1984; van der Ende et al ., 1985; Ogawa et al ., 1985). Par-ticipation of RNA polymera se in the initiation of replicationis also indicated by ear lier f indings (Zyskind et al ., 1977;Ful ler et al ., 19 81).

In response to mutational changes in or iC, the soloprimase syste m (Figure 12A) shows a repl ication patternsimi lar to that observed for formation of the prepr imingcomplex. Deletion of the left 19mer greatly reduces repli -cation ac tivi ty; fur ther deletion of 4 bp of the middle 13-merabol ishes repl ication. On the other hand, m utations at theright end of oriC cause l i tt le reduction. By contrast, repli -

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(A) Solo Primase

0 30 60 90 120dnaA PROTEIN (ng)

Figure 12. In Vitro Replication of Mutant oriC PlasmiosReactions (25 p l) contain ing supercoiled p lasmid DNA (75 fmol) and dnaA prote in as indicated were incubated for 20 min at 30%. (A) Solo primasereconstituted enzyme system. (Et) RNA polymeras e-dependent reconstituted enzyme system. (C) dnaA mutan t fraction II system. (See Exper imenta lProcedures.)

cation in the RNA polymerase-dependent syste m (Figure126) resembles the behavior of the mutants in vivo. Dele-t ion of even one 13-mer abol ishes the capacity of the plas-mid to serve as a template. Even mutations at the r ight endof 0% enfeeble the plasmid in this syste m. Presumably,the higher levels of HU protein that lead to the requirementfor RNA polymerase activation also contr ibute to a morestr ingent requirement for the intact or iC sequence. At thehigher H U concentrations, more dnaA protein is also re-quired to activate the or igin. In a crude enzyme fraction(Ful ler and Kornberg, 1983), much as in the RNA polymer-ase-dependent reconsti tuted syste m, repl ication of mu-tated templates (or iCAQR, or iCVQR, and ofiCA13L) isgreatly affected (Figure 12C). These results, so simi lar tothose observed in vivo, suggest that both the crude en-zyme fraction and the RNA polymerase-dependent re-consti tuted syste m closely reflect the cel lular co ndit ions.DiscussionBy analyzing deletions in or iC and using Pl nuclease asa probe for single-stranded DNA , we have demonstratedthat the duplex is opened by the action o f dnaA protein ator iC and is maintained in an even m ore stable open formby the prepr iming complex. Ear l ier mutational studies as-sessed or igin function only in vivo (Oka et al ., 1982, 1984)and were limited essen tially to an all-or-none result. Theparallel in vi tro studies presented here al low a much m orerefined analysis of al tered or igins. Deletions in the 19merregion we re readily obtained by exploiting the 13-mer-specif ic cleavage of the open com plex by Pl nuclease,and the si tes of cleavage by certain restr ict ion nucleases.The stage at which each mu tation blocks init iation hasbeen determined, and the residual levels of activi ty a t thisand subsequent steps have been measured.

The possibi li ty remains that the DNA does not becomesingle-stranded, but takes on some other conformationthat is Pl-sensi t ive. Howeve r, bending or kinking of theDNA is inconsistent with the observed dnaAdependentP l cutt ing of opposite strands within one or two bases.

Bending could enhance attack only on one strand at anypoint. Th at the structure is not Z-DNA is shown by the coldsensit iv i ty of the open complex. The simplest explanationis that the 13-mer DN A is indeed single-stranded in theopen complex. This is consistent with the documentedspecif ic i ty of Pl nuclease (Kowalski , 1984) the suscepti-bi l i ty of both strands to Pl attack, the requirement for hightemperature and sensit iv i ty to cold, and also the avai labi l-i ty of the template for subsequent dnaB hel icase action.

The 13-mer SequenceAt the left end of or iC, three 13-mers in tandem have beenidentified, coinciding with the site for duplex opening bythe dnaA prote in in the initiation of replication. This sug-gests that dnaA protein speci f ically recognizes these13-mer sequences. Data supporting this idea include thefol lowing: First, two such 13-mers are located close to thednaA box in the origin o f a plasmid (pSC101 ) also depen-dent on dnaA protein. Second, the 13-mer sequence is al-mos t as highly conserved among or iC sequences in dis-tantly related E nterobacteriaceae as are the dnaA b oxes,the only deviations being in the sixth and ninth posi t ions,the two nonspecif ic posi t ions in the consensu s. Third, de-letion of ei ther the leftmost or part of the middle 13-merinactivates or igin function in vivo, whereas an insertion of4 bp between them is tolerated (Oka et al ., 1980). Fou rth,deletion mutants of or iC lacking the left and middle 13merscan sti l l form an open complex that is sensi t ive to Plnuclea se, sp ecifically in the remaining (right) 13-me r,whi le removal of al l three 13-mers abol ishes duplex open-ing. Fi fth, many other prokaryotic or igins possess equiva-lent tande m repeats in their AT-rich regions; these originsinclude those of h, @80, @82, Pl , F, Rl, R8K, R K2, and P4,as well as the putative Bacillus subtil is oriC (Bramhill andKornberg, unpublished data).A Model for OK InitiationThe insights provided by probing with the single-strand-specif ic Pl nuclease enable us to refine our model of theear ly ini tiat ion events at or iC. We propose the fol lowing

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Figure 13. A Scheme for oriC In it ia tionDetails are g iven In the text. The 1 to 1% nega-tive turns of oriC DNA wrapped about the dnaAprotein core is suggested from earlier work(Fuller and Kornberg, 1983; Fuller et a l., 1984;Funnell et a l., 1987).

SUPERCOILEOTEMPLATE AND -REPLICATION

stages (when HU protein at a stimulatory level coats about3% of the plasmid) (Figure 13): First, dnaA protein bindsto i ts 9 bp boxes in OK. Second, an ini tial dnaA-or iC com-plex is formed, incorporating 20-40 monome rs of dnaA ina central core around which or iC DNA is wrapped. Third,stepwise duplex opening of the 13-mers by dnaA protein oc-curs at an elevated temperature to form an open c omplex.Fourth, the dnaB and dnaC proteins asso ciate (probablyas a B-C complex) with the open com plex by protein-pro-tein interactions. Fi fth, the dnaB he l icase enters to pro-duce a prepr iming complex; the hel icase probably recog-nizes the single-stranded DNA structure, a potential fo rk,rather than the sequence of the 13-mers from which i tprobably displaces dnaA protein. Further dnaB hel icaseaction, upon addit ion of SSB and gyrase, unwinds thetemplate and exposes i t to action by pr imase. Guided bydnaB, primase may synthesize RNA primers tha t DNApolymerase I l l holoenzyme can extend.Open Complex FormationOpen complex formation requires only the dnaA protein,as indicated by the copur i f ication of the duplex-openingactiv ity with the replication-initiation activ ity of the nearlyhomogeneous dnaA protein, and the persistence of the Plnuclease cutt ing pattern in the 13-mers in the presence ofonly the dnaA protein (omitt ing IHF and HU proteins).

Retention of only one of the three 13-mers suff ices forthe formation of a Pl-sensi t ive open com plex at 38%. Al-though the deletions oriCAl3L and oriCAl3LM result inequal ly AT-r ich contiguous DNA sequences replacing thedeleted DNA , mapping reveals tha t the Pl cuts l ie almostexclusively in the remaining 13-mers and not in these ad-jacent AT-rich sequences. These results suggest that dnaAprotein s pecifica lly recogn izes all or part of the 19mer se-quence. Since each 13-mer includes a dam methylationsequence, specif ic recognition by dnaA protein of one orboth of the modified adenines in the 13-mer could accountfor the observed effects of dam methylation in vivo and invi tro (Messer et al ., 1985; Smith et al ., 1985). A simple ex-per imental approach to defining the precise sequence re-quiremen t for dnaA p rotein recognition would b e to ana-lyze point mutations in a single 13-mer for open complexformation by the dnaA-dependent Pl cutt ing assay.

COMPLEX

The si te-specif ic duplex opening activi ty of dnaA proteinimpl ies that dnaA may be able to recognize two distinctDNA sequences. One is the 9 bp dnaA box, as a duplex,and the other the 19m er as either double- or single-stranded DNA . A simi lar dual sequence specif ic i ty hasbeen found in the actions of phage lambda si te-specif icint recombinase (Ross and Landy, 1982, 1983). The int en-zyme fi rst binds three specif ic arm si tes in a supercoi leddonor D NA substrate, organizes the DNA around a centralcore of protein (Richet et al ., 1986), and then recognizesin both the donor and acceptor molecules a second junc-t ion sequence that serves as the si te for strand exchange.

Simi lari t ies can also be seen between the 13-mers andpromoters for RNA polymerase, both at the structuraland functional levels. Like a transcr iptional promoter, the13-mers in or iC serve as the entry si te for an enzyme toinvade a duplex DNA region. Both types of entry se-quence are AT-r ich, thereby faci l itat ing strand separation.Sharp temperature transi t ions are observed in the forma-tion of open complexes by RNA polymerase and by dnaAprotein.

Each 13-mer opened wi l l lead to the loss of sl ightly morethan one superhel ical turn per template. Thus, successiveopening events w i l l require more energy. A considerationof the physical constraints imposed by the dnaA proteincomplex binding t ightly to the adjacent port ion of or iC sug-gests that the opening events m ay be sequential , start ingwith the r ightmost 13-mer, nearest the 9 bp dnaA box (Fig-ure 14). Such a stepwise mechan ism avoids several prob-lems of a one-step mechanism by requir ing recognition ofonly a short D NA sequence such as can be achieved bya helix-turn-helix or an equivalent protein dom ain, andlower activation energies fo r individual opening eve nts.This stepwise mechan ism also faci l i tates opening up justover one hel ical turn at a t ime and real igning the DNA forthe next opening step.The nucleotide requirements for dnaA-dependent opencomplex formation are rather compl icated. Sti l l, our obser-vations might be explained by a single ATP si te per dnaAmolecule. Of the 20 or more dnaA subunits in the opencomplex, some may bind the 9-mers and some the Wmers,some interact with the dnaBC complex, whi le yet othersmay only contact adjacent monom ers. Such var ied inter-

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) dnaAA

lll l l II,

) dnaA

Figure 14. Proposed Sequentia l Opening of the 13-mers by dnaA Pro-te in and Subsequent Entry of the dnaB-dnaC ComplexThe 13-mers are indicated by arrows, the leftmost 9-mer by a stippledbox (cf. Figure 8).

action s could result in significan t differenc es in affinitiesfor ATP and i ts functions.Three distinct requirements for ATP in formation of anopen complex are impl ied by three apparent K, values:The f i rst is a very t ight binding of ATP and also of ADP,CTP , dATP, and ATP yS, with a KD of 30 nM (Sekimizu etal ., 1987). ATP activates dnaA protein for repl ication, whi leADP b locks strand opening, even in the presence of 5 mMATP; release of the bound nucleotide is very slow ( t l ,Z of30 min). Hydrolysis of the t ightly bound ATP to ADP isequal ly slow. Inasmuch as t ightly bound ATPyS can alsoactivate dnaA protein for strand opening, in the presenceof 5 mM ATP , an alloster ic role for this interaction with ATPseems l ikely. A second requirement for ATP has a K,near 1 PM. Although CTP and dATP substi tute for ATP,some properties distinguish this interaction from the high-aff ini ty interaction. Analogs such as ATPyS, AM PPNP,and AMPPCP are inactive but appear to inhibit competi-t ively. The need for a hydrolyzable nucleotide impl ies thatin this case ATP m ay provide energy ei ther for strandopening or for helicase entry. A third requirement for ATPhas a low aff ini ty, with a K,,, around 1 mM . As in the othertwo cases above, dATP and CTP can substi tute for ATPAnalogs such a s ATPyS and AMPPCP are also effectivehere, suggesting a second al losteric interaction of dnaAprotein with ATP

That the same set of nucleoside tr iphosphates (ATP,CTP , dATP) is effective in each case, and tha t no morethan a single ATP molecule is bound per dnaA subunit innitrocellulose filter binding (Sekim izu et al., 1987), arguesin support of a single si te. This model also reconciles therequirement for a hydrolyzable nucleotide for open com-plex formation (K, < 1 PM) with the very low level of ATPhydrolysis observed dur ing prepr iming complex formation(Sekimizu et al ., 1987), since i t explains why hydrolysis ofATP might occur on only one or a few crucial subunits ofdnaA in the complex. Such an essential hydrolysis of ATP

may also provide a key to regulation: production of inac-t ive ADP-dnaA protein within the complex coupled todnaB helicase entry would insure a single initiation perorigin.Prepr iming Complex FormationThe Pl-sensi t ive structure formed by dnaA protein at 38%in the presence of ATP is unstable at 16%. To produce acomplex that retains a Pl-sensi t ive configuration at 16%,dnaB and dnaC prote ins are both required during the38% incubation to form a prepr iming complex. Thus,dnaB hel icase or dnaC, rather than dnaA protein, appearsto be responsible for maintaining the single-stranded re-gion in the prepriming com plex. Such a role for dnaBprotein can explain the difference in the Pl-sensitive sitesbetween the dnaA open complex and the prepr iming com-plex. Protection of the prepr iming complex from Bgll l re-str ict ion nuclease cleavage also supports direct dnaB ordnaC involvement and demonstrates that the prepr imingcomplex includes the 13-mer region. Th is is consistentwith the ear lier f indings by electron microscopy (Funnel let al ., 1987) that app roximately 50 bp of the left end of or iCis included in the prepriming com plex, but not in the ini-t ial complex with dnaA protein alone.

Eff icient formation of a stable prepr iming complex re-quies al l three 13-mers and an elevated temperature.These observations suggest that whi le the sensit iv i ty toPl of the open c omplex requires that only one 18mer berecognized by dnaA protein, eff ic ient e ntry of dnaB hel i-case depends on having al l three 13-mers opened. Thisproposal is reasonable in view of the larg$ size of the hex-americ dnaB (300 kd; Stpkes radius, 58 A) or dnaBC (480kd; Stokes radius, 64 A) complexes (Kobor i and Korn-berg, 1982). The higher temp erature required for formingthe prepr iming complex is also consistent with this model.

The formation of a prepr iming complex exhibi ts anucleotide requirement in addit ion to that for open com-plex formation. Only ATP is able to support prepr imingcomplex formation, whereas CTP and dATP are both ef-fective for duplex opening. If there is in fact only a singlesi te for ATP (shared by CTP and dATP) per dnaA mono-mer, then this absolute requirement for ATP to form aprepr iming complex most l ikely reflects an interaction withdnaB or dnaC proteins, as in the formation of a dnaB-dnaC complex (Wickner and Hurwitz, 1975; Kobor i andKornberg, 1982).Man y other origins have an organization similar to oriC,including those of lambdoid phages, certain broad host-range plasmids, and even SV40. The SV40 T antigenleaves a footpr int on portions of the SV40 minimal or i-gin (Tjian, 1978), but not on an essen tial AT-rich region(Stil lman et al ., 1985). The T antigen also possess es ahel icase activi ty (Stahl et al ., 1986; Dean et al ., 1987). Themode l based on the actions of dnaA protein in initiatingoriC ma y prove to be applicable to initiation of a widerange of or igins, prokaryotic and eukaryotic.Experimental ProceduresReagentsATP. deoxyribonucleoside tr iphosphates, and HEP ES were from Phar-

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mac ia ; GTR CTP UTP and ADP were f rom Sigma ; ATPTS. AMPPNP ,and AMP PCP were from Boehringer; [Y-~~P]ATP (7000 Cilmmol) wasfrom New England Nuclear; [a-32P]dATP (3000 Cilmmo l) was fromAmersham.Enzymes and Prote insSamples of purif ied E. coli IHF were g ifts from H. Nash (National Insti-tutes of Health) an d N. Cozzarelli (University of Califo rnia, Berkeley);both preparations are essentia lly free of contaminating HU prote in asjudged by SDS-polyacrylamide gel analysis. Purif ied replication pro-teins were prepared as described by Kag uni and Kornberg (1984). ThednaA prote in was purif ied by the method of Fuller and Kornberg (1983)to a specific activity of 1.0 x 106 Ulm g and a purity greater than 90%as judge d by SDS- polyacrylam ide gel electrophoresis. Restriction en-zymes, T4 polynucleotide k inase. T4 DNA ligase, and Sl nucleasewere from New England Bio labs; calf in testinal phosphatase was fromBoehringer; Pl nuclease was from Pharmacia.Bacteria l Stra insE. coli stra ins MM383 @o/A72 lacZ53 rpsLl51 thyA rba-5 deoC2IN(rmD-rrnE)l) and MM384 (isogenic pa/A+ control) (Monk and Kin-ross, 1972) were used for testing oriC function in vivo. Strain DHl (recA1endA hsdFfl7gyrA96supE44 fb i-1) (Hanahan, 1983) was used for mu-tant constructions.Construction of Mutant oriC PlasmidsPlasmid pBSoriC contains a 678 bp Hincll-Pstl fragment spanning oriC(-189 or +489) cloned i nto the pBluescrip t vector (Stratagene, Inc.,San Diego) (T Baker and A. Kornberg. unpublished data). The Bgllls ites at the left end of oriC allow the specific deletion of the leftmost13-mer (pBSoriCA13L) or, fo llowing Sl nuclease tr imming, the left and4 bp of the middle 13-mer (pBSoriCA13LM). More extensive deletionsof the left end of oriC were constructed by explo it ing the Pl nucleasesensitiv ity induced by dnaA prote in. Pl- l inearized pBSoriC DNA mole-cules were recovered from a 0.7% low melting point agarose gel (Sea-plaque), restricted with Bgll l, and isolated from a second low meltingpoint gel run as almost full-le ngth linear species free from shorterDNAs. Religation generated a series of deletions. The shortest dele-tion, designated pBSoriCA 13LMR lacked the leftmost 43 bp of oriC.The Hindll l-Accl fragment was deleted to produce pB SoriCAS R, and4 bp insertion at the Hindll l s ite (pBSoriCV9R) was produced by fi l l ingin the Hin dlll sticky ends by DNA polymerase I. In each case, reli gationwas performed at 10 uglml DNA to favor recircularization. pCM959(Meijer et a l., 1979) a g ift from M. Meijer (Amsterdam), is a 4012 bpminichromosome contain ing oriC (-677 to +3335). P lasmid DNAswere prepare d as described by Ogawa et al. (1985).Pl L inearization of Open ComplexesThe standard reaction (50 p l) contained 40 mM HEPES -KOH (pH 7.6)8 mM magnesium acetate, 30% (vol/vol) g lycerol, 320 pglml B SA, 150fmol of supercoiled p lasmid DNA, 67 ng of HU prote in, 240 ng of dnaAprote in, and 5 mM ATP Follow ing incubation at 38% for 2 min, 1.2 Uof Pl nuclease was added in 3 u l of 30 mM potassium acetate (pH 4.8).Pl c leavage was stopped after 5 set by the addition of 40 u l of 25 mMEDTA. 1% SDS. For quantitation of the fraction of l inear molecules, aportion of the reaction was electrophoresed through a 0.7% agarosegel in 100 mM Tris-borate (pH 8.3) 1 mM EDTA (TBE) at 6 V/cm, sta inedwith eth id ium bromide, and photographed using Polaroid f i lm . Densito-metric scanning of the negative was used to determine the proportionof l inear molecules.Pl L inearization of Preprimlng ComplexesReactions, in 50 u l, were s imilar to the open complex reactions exceptdnaB (120 ng) and dnaC (40 ng) were included, and the free mag-nesium concentration was adjusted to 0.3 mM by addition of EDTA tochelate excess magnes ium contributed by theenzyme buffers. Incuba-tion at 38% was for 30 min, after which reactions were chil led to 16%and Pl nuclease (1.2 U) was added. After 5 or 10 sec. d igestion wasstopped by the addition of EDTA and S DS as for open complexes.Quantitation was also by identical methods.Mapp ing P l Cut S i tesLinearized DNAs were extracted with phenol-chloroform, precip itated

with ethanol, and washed with 70% ethanol. F ollowing dephosphoryla-

tion and 5 end-labeling with [y-s*P]ATP using T4 polynucleotide k i-nase, DNAs were restricted with appropriat e enzymes and preparedfor electrophoresis. To obtain nucleotid e-level resolution, urea-poly-acrylamide sequencing gels (60 cm x 0.2 mm) were run on an LKB2010 Macrophor apparatus at 65C 2300 V for 2-4 hr in TBE buffer.Maxam-Gilbert sequencing reactions, DNAase I d igestion ladders,and Bgll l restriction fragments recutwith the appropriate enzyme wereused as standards to locate the positions of the PI cuts.In Vitro Replication AssaysThe fo llowing methods have all been described previously: RNA poly-merase-d ependent reconstituted replicatio n assays (Ogawa et al.,1985); solo primase reconstituted replicatio n assays (van der Ende eta l., 1985); and dnaA mutant fraction II complementation assays (Fullerand Kornberg, 1983).AcknowledgmentsWe are grateful to L. Bertsch for careful reading of the manuscript, andto D. Brutla g for advice a nd assistance in computer searches. This re-search was supported by grants from the Natio nal Institutes of Healt hand the National Science Foundation.

The costs of publica tion of this article were defrayed in part by thepayment of page charges. This article must therefore be herebymarked advertisement in accordance with 18 U.S.C. Section 1734solely t o indicate this fact.Received October 13, 1987; revised Dec ember 31, 1987.

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