Cell, Vol.

52, 743-755,

March

11, 1988, Copyright

0 1988 by Cell Press

Duplex Opening by dnaA Protein at Novel Sequences in Initiation of Replication at the Origin of the E. coli Chromosome
David Bramhill and Arthur Kornberg Department of Biochemistry Stanford University School of Medicine Stanford, California 94305 templates to be replicated (Funnell et al., 1986). In vitro, a sharp increase in the rate of prepriming complex formation is observed at around 30% as the temperature is increased (van der Ende et al., 1985). No direct evidence has previously been obtained to indicate that any part of the template becomes singlestranded at very early stages prior to dnaB helicase action. Here we identify a 13 bp sequence present as three repeats in oriC, and demonstrate that these sequences are opened in the prepriming complex. With the singlestrand-specific nuclease Pl from Penicillium citrinum (Kowalski, 1964; Camilloni et al., 1986) as a probe, we show that the initial duplex opening occurs within these repeats, and is catalyzed by the dnaA initiator protein. We also report the construction of deletion derivatives of oriC and their activity in each step of initiation. The results strongly support the proposal that dnaA protein recognizes the 13-mer sequences and, furthermore, suggest a mechanism of successive opening of all three 13mers preparatory to dnaB entry. Results Three 13 Base Tandem Repeats in oriC The boundary at the right end of oriC (Oka et al., 1980) coincides with the end of the rightmost of four 9 bp sequences (boxes) bound by dnaA protein (Figure 1). The leftmost box is about 60 bp from the left boundary of oriC. Thus, the absolute requirement for the dnaA protein in initiation in vivo and in vitro (Fuller and Kornberg, 1983) can account for the role of the righthand segment of oriC, but not of the leftmost 60 bases. Comparison of the oriC sequences of the Enterobacteriaceae (Zyskind et al., 1983) reveals that this leftmost sequence is highly conserved and contains three tandem repeats of a 13 bp motif (Table 1); the consensus sequence indicates that 11 of the 13 positions are highly conserved. Sequences fitting this consensus also occur as two tandem repeats at a distance of 7 bp from the dnaA box within the origin of plasmid pSC101; as with oriC, initiation at the pSClO1 origin requires dnaA protein (Hasunuma and Sekiguchi, 1977; Frey et al., 1979; Felton and Wright, 1979). Computeraided search of sequence data bases of E. coli and its phages and plasmids revealed no other tandem repeats of this 13 base consensus other than those in Table 1. Of the few single close matches identified, two are noteworthy. One occurs in the dnaA gene promoter (Hansen et al., 1982) only 17 bp from a dnaA box (Fuller et al., 1984); the other is near the 16 kd gene promoter, 10 bp from its dnaA box (Buhk and Messer, 1983). Consistent with the consensus deduced from the E. coli sequences, the few differences between the left ends of Enterobacteriaceae OriC sequences occur almost exclusively at either of the two nonspecific positions in the sequence. Deletion of either the left 13-mer or part of the middle one inactivates oriC; a C-T change at the fourth base of the middle 13-mer reduces oricfunction. No effect

Summary Three tandem repeats of a 13-mer in the AT-rich region are essential to the unique replication origin of E. coli and of remotely related Enterobacteriaceae. These iterated sequences are identified by deletion analysis and sensitivities to endonucleases as the site for initial duplex opening by the initiator dnaA protein. This open complex requires ATP and 38% for optimum formation and stability. The subsequent dnaCdependent entry of dnaB helicase to form a prepriming complex stabilizes the open structure, blocks cleavages by a restriction endonuclease in the 13-mer region, and broadens the endonuclease cutting pattern. We propose that dnaA protein recognizes and successively opens the 13-mer sequences, thereby guiding the entry of dnaB helicase into the duplex preparatory to priming of replication. Introduction In Escherichia coli, a unique sequence, oriC, serves as the site for the initiation of chromosomal replication during normal growth. The functional elements of oriC are contained within a 245 bp sequence highly conserved among the Enterobacteriaceae (Oka et al., 1980; Zyskind et al., 1983). Four 9 bp sequences (dnaA boxes) bind the initiator dnaA protein (Fuller et al., 1984). However, the function of a 50 bp region at the left end of oriC, also strikingly conserved, has not been identified. Using purified enzymes to replicate supercoiled oriCcontaining templates, the sequence of events preceding DNA synthesis has been divided into four stages: first, recognition of oriC by dnaA protein and cooperative binding to the four dnaA boxes; second, incorporation of dnaB and dnaC proteins into a prepriming complex; third, bidirectional unwinding by the dnaB helicase to generate two forks; and fourth, priming by dnaG primase for chain elongation by DNA polymerase Ill holoenzyme (Kaguni and Kornberg, 1984; Ogawa et al., 1985; van der Ende et al., 1985; Baker et al., 1986, 1987; Funnell et al., 1987). Protein HU stimulates the early stages (Ogawa et al., 1985), and single-strand binding protein (SSB) and gyrase must be present during opening of the duplex to stabilize the single-stranded DNA and relieve the torsional stress (van der Ende et al., 1985; Baker et al., 1986, 1987). Negatively supercoiled DNA has a greater affinity for dnaA protein (Fuller and Kornberg, 1983) and is required for oriC
Present University, address: Stanford, Department of Biological California 94305. Sciences, Stanford

Cell 744

minimal
13 mers psc101 ori

origln~

16 kd gene

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

Figure 1. Locations

of the 13-mer

Repeats Figure 2. Pl Nuclease dnaA Protein

The diagram indicates 13-mers (left [L], middle [M]. right [R] by large solid arrows, 9-mer dnaA boxes by stippled boxes, RNA polymerase promoters by lightly stippled arrows, and pSClO1 rep protein binding sequences by small arrows. The figure is not drawn to scale.

Linearization

of an oriC Plasmid

Depends

on

The reactions, containing 150 fmol of pCM959 supercoils in 50 ul were as described in Experimental Procedures. Open complex formation and Pl nuclease probing were at 38%.

is observed when a 4 base insertion is made between the left and middle 13-mers (Oka et al., 1980). Probings with Endonucleases Opening of the Duplex Is Catalyzed by dnaA Protein Funnell et al. (1987) observed that the 13-mer region of oriC is involved in the prepriming complex but not in an early oriC-dnaA complex. The sharp transition in temperature profile (van der Ende et al., 1985) and the requirement for a negatively supercoiled template for prepriming complex formation (Funnell et al., 1986) both suggest that the initial opening of the duplex occurs at an early stage. The AT-rich nature of the 13-mers suggests a possible role in melting the duplex DNA. To test this directly, Pl endonuclease was used to probe initiation intermediates. This nuclease, similar to Sl in its specificity for cleavage of single strands (Kowalski, 1984) has the advantage of acting at pH 7.6, the optimum for oriC replication. Action by dnaA protein sensitizes oriC-containing supercoils in the 19mer region to linearization by Pl endonuclease. Two types of Pl-sensitive complexes can be

distinguished: an open complex, which requires only dnaA protein for its formation and a high temperature (e.g., 30%) for both its formation and its stability; and the prepriming complex, which requires dnaB and dnaC proteins in addition to dnaA protein for formation, but which, once formed at a high temperature, is stable to chilling to 16% or even to 0C. Several characteristics of the open complex indicate that it is a true intermediate in initiation: first, the need for high temperature; second, the requirement for dnaA protein levels of 20-40 monomers per ofiC sequence (Figure 2), similar to those in the overall reaction (Fuller and Kornberg, 1983); third, the extent of Pl sensitivity (up to 50% of the input DNA), suggesting that it is not a minor byproduct; fourth, the requirement for the oriC sequence (see below); and fifth, the inactivity of the ADP form of dnaA protein in open complex formation, despite the addition of ATP to 5 mM (Sekimizu et al., 1987; and see below). Inclusion of HU protein greatly reduces the background level of nonspecific cutting by Pl, but its known stimula-

Table

1. Thirteen

Base Pair Repeats

and Deduced

Consensus

Sequence Distance from dnaA Match 10111 10111 9111 11111 9/l 1 9111 9/l 1

Site oKa

Location L M R L R

Sequence

(5 to 31

Box (W

GATCTATTTATTT GATCTGTTCTATT GATCTCTTATTAG GATCTATTCTTTT TATCTTTTTTTAT GATCTTCTGTTTC GATCGGCTTTTTT GATCTnTTnTTTT

13 7 17 10

oripSClOl~ dnaA promoterC 16 kd gene promote? Consensus

The 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 the sequence given here. The predominant or only base occurring at a given position is indicated in the consensus; where no particular base predominates, n is used, The number of matches to the 11 specific positions in the 13-mer consensus is indicated, as is the distance from the nearest dnaA box. a Meijer et al., 1979; Oka et al., 1980. b Vocke and Bastia, 1983; Churchward et al., 1983; Armstrong et al., 1984; Yamaguchi and Yamaguchi, 1984. c Hansen et al.. 1982.

dnaA-Catalyzed 745

Duplex

Opening

Haalll
-II

Aval

(c) &qGoG q?G-jGq~GoG ,&

(A)
dnaA --@@--@@ 9-mer u

UN
dnaA

Hindlll r oc @ WC @ -

1
W
SnaBl -& q9

pCM959

EcoRV & ,j9

IL- R T
- LINEAR \ 13.mers

m 13.mars a

- 13.mers

Figure

3. Mapping

Pl Cleavage

Sites to the 13-mer

Region

of OX

Open complexes (OCs) or prepriming complexes (PPCs) were formed as described in Experimental Procedures in B-fold reactions, each containing 750 fmol of pCM959. Controls lacking dnaA protein were also probed with Pi nuclease. DNAs were dephosphorylated and end-labeled using T4 polynucleotide kinase before restriction analysis. (A) Location of the relevant restriction enzyme sites in pCM959 DNA near oriC. (B) Native gel showing that the predominant cuts lie near the left end of ofiC. Samples of Pl-linearized. 5end-labeled DNAs were restricted with either SnaBl or EcoRV, electrophoresed in a 1.2% agarose gel, and autoradiographed. The positions of the Bglll(13-mer) sites in oriC are indicated for each case, determined by comparison with double digests (not shown). (C) Mapping cleavage sites on the lower strand with respect to the Aval and Haelll sites. Samples of PI-linearized, 5 end-labeled DNAs were restricted with either Haelll or Aval, electrophoresed in an 8% polyacrylamide denaturing gel, and autoradiographed. The positions of the 18mers relative to the Aval site are indicated by the solid arrows (oriented as in Figure 1). The Aval fragments are 1 base longer than the Haelll fragments. (D) Mapping Pl cleavage sites in the upper strand. Samples of PI-linearized, 5 end-labeled DNAs were restricted with Hindlll and electrophoresed on a 6% polyacrylamide denaturing gel. The solid arrows (oriented as in Figure 1) indicate the locations of the Id-mers. The lane of the sample of open complex plus dnaA was exposed for only 1 day; the other lanes were exposed for 4 days.

tory effects on initiation (Dixon and Kornberg, 1983) are difficult to measure by Pl sensitivity. The Sites Cut by PI N&ease Lie in the Id-mers To determine the location of the sites cut by Pl, the unique restriction targets for SnaBl and EcoRV in pCM959 were used as reference points (Figure 3A). Following dephosphorylation, the 5 ends generated by Pl linearization were labeled with 32P by T4 polynucleotide kinase. DNAs were then restricted with either SnaBl or EcoRV, electrophoresed in a 1.2% agarose gel, and autoradiographed (Figure 38). The great majority of the sites cut by Pl are clustered together at the left end of oriC, near the Bglll sites. No significant linearization was detected at any other point in or near oriC. The full-length linear molecules presumably result from labeling at nicks introduced by Pl nuclease, since they are observed even following overdigestion with the appropriate restriction nuclease. They are most likely distributed throughout the templates. More precise mapping of the predominant sites was ob-

tained by using Aval, Haelll, and Hindlll restriction sites located close to the 13-mers in oriC (Figure 3A) and analysis on denaturing polyacrylamide gels. The locations of the major sites of Pl cleavage are all within the 19mer sequences (Figures 3C and 3D). The cut sites in the open complex are predominantly clustered in the middle 13-mer on the upper strand, yet widely distributed in each 13-mer on the lower strand. This may reflect a protection of the upper strand by dnaA protein. dnaA Protein A/one Confers PI Sensitivity to the It-mers That the specificity for the AT-rich 13-mer regions should depend on recognition by dnaA protein is suggested by their proximity to a dnaA box and also by the absence of 13-mers from origins that require proteins other than dnaA (Bramhill and Kornberg, submitted). The possibility that a contaminant activity in the dnaA protein preparation might contribute to recognition of the 13-mer sequences has been removed by an improved purification procedure,

Cell 746

Table 2. IHF Can Replace Open Complex Formation Additions HU and dnaA HUa IHF and dnaA lHFa

HU Protein

for

dnaA HU

-@@@@--

Linear 47 9 52 10

Molecules

(o/o)

Reactions were as described in Experimental Procedures. Protein additions, where indicated, were: dnaA, 240 ng; HU, 67 ng; IHF, 60 ng. a When neither IHF nor HU is present, a high level (40%) of dnaAindependent linearization is observed. This background cutting is distributed around the plasmid and is not localized to the 19mer region (see rightmost lane, Figure 4).

L I 13.merr

which yields dnaA protein containing no detectable impurities (Sekimizu et al., submitted); both the Pl-sensitizing and replication activities copurify on FPLC (Pharmacia) gel filtration and Mono-S ion exchange chromatography (data not shown). In addition, integration host factor of E. coli (IHF) (Nash and Robertson, 1981) can replace the HU protein at similar levels (Table 2) without altering the overall extent of the dnaA-dependent reaction. These two similar, basic proteins are interchangeable in reducing the nonspecific Pl background cutting. The fact that they support the dnaAdependent linearization with similar efficiencies suggests that neither contributes to the sequence specificity. The dramatic effect of low levels of HU or IHF (sufficient to coat only 3% to 5% of the template) in reducing the nonspecific cutting reaction by Pl nuclease indicates the profound conformational changes induced by low levels of either protein. IHF also substitutes for HU protein in the stimulation of the reconstituted replication of oriC templates (D. Bramhill, T A. Baker, and A. Kornberg, unpublished data). Finally, the major cleavage sites for Pl endonuclease induced by dnaA protein in the presence of HU are identical to those induced by dnaA protein alone (Figure 4). In the latter case, the extent of 13-mer-specific cutting is only one-fifth that observed in the presence of HU or IHF. In addition, in the absence of HU and IHF, minor dnaA protein-independent Pl cleavage sites are also detectable outside the 13-mer region of 0% (not shown). Nucleotide Requirements for Opening of the Duplex The dnaA protein has a very high affinity (Ko = 30 nM) for ATP, ATPyS, and ADF, with which it forms very stable complexes (Sekimizu et al., 1987). Both the stability of dnaA protein and its activity are profoundly affected by nucleotide binding: the ATP and ATPyS forms are both active in replication (in the presence of 5 mM ATP), whereas the ADP form is inactive and fails to induce a Pl-sensitive structure in oriC (Sekimizu et al., 1987). In addition to the requirement for micromolar levels of ATP open complex formation requires a level of ATP near 5 mM for optimum efficiency. Both dATP and CTP can substitute for ATP in open complex formation (Table 3) but GTP UTP, dTTP, and dCTP failed to support duplex opening (data not shown). The ATP analogs ATP$S, AMPPNP, and AMPPCP at 5 mM cannot replace ATP completely.

Figure 4. Comparison of Pl Cleavage Sites in oriC Induced Protein in the Presence or Absence of HU Protein

by dnaA

Reactions containing 150 fmol of pCM959 DNA were incubated at 36OC for 2 min with 67 ng of HU protein or 240 ng of dnaA protein or both or neither, as indicated. Linearization with 1.2 U of Pl nuclease for 5 set was at 38% Following phenol extraction, samples were 5 end-labeled and restricted with Hindlll. Electrophoresis was on a 8% polyacrylamide denaturing gel. The locations and orientation of the 19mers are indicated.

Table 3. Nucleotide Requirements Complex and Prepriming Complex

for Formation

of Open

Open Nucleotide ATP CTP dATP None

Complex

(%) Minus dnaA 6 7 10 10

Prepriming Complex (o/o) Full Reaction 85 5 4 5 Minus dnaC 10 6 5 5

Full Reaction 53 50 46 8

The dnaA protein was first incubated with 1 uM ATP for 15 min at 0% to generate the ATP form of dnaA protein. Complexes were formed at 38% as described in Experimental Procedures, with the indicated nucleotide present at 5 mM. Open complexes were probed with Pl nuclease for 5 set at 38%; prepriming complexes were chilled to 16% and then probed with Pl nuclease for 5 sec. The fraction of molecules linearized by Pl was determined and is expressed as a percentage.

$A-Catalyzed

Duplex

Opening

Table 4. Requirements Prepriming Complex Component % 8 ; CL P d 2 z g L

x i 15

for Formation

of a PI-Sensitive

Omitted

Linear 95 IO a 12 62

Molecules

(%)

40 35 30 25 20

plus

5 mM AMP-PCP

None dnaA dnaB dnaC HU None;

no 36OC preincubationa

Reactions, containing 150 fmol of pCM959 supercoils, were as described in Experimental Procedures. Components were omitted as indicated. 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 min before Pl probing. \ -..-. I 2 ATP aone I I 3 4 LINEARSICATPI I I 5 6 (percent-PM.

g 0

IO 5

I 1 dnaA-DEPENDENT

) of

Figure 5. Eadie-Hofstee the Open Complex

Plot of Dependence

on ATP of Formation

Reactions were as described in Experimental Procedures. The ATP concentration was varied from 0 to 5 mM; in one titration AMPPCP was included at 5 mM. The proportion of molecules linearized by PI nuclease was determined, and the dnaA protein-independent background (6%) was subtracted. Production of dnaA-dependent linear molecules was a measure of the initial rate.

However, in the presence of these analogs, much lower levels of ATP can activate the reaction. Assuming that the extent of dnaA-dependent Pl linearization is proportional to the initial rate of duplex opening, more refined kinetic analysis is possible. This assumption is consistent with the observed time course for the duplex opening reaction, which shows a 5 set lag followed by a rapid approach to the maximum Pl sensitivity. In the presence of 5 mM ATP the increase is more rapid (t,/, 2 4 set) than at lower ATP concentrations (T,,, 4 8 set at 1 mM ATP) (data not shown). Analysis of these data by a Lineweaver-Burke (not shown) or Eadie-Hofstee plot (Figure 5) indicates two distinct K, values for ATP: one high-affinity interaction (K, < 1 PM) and one low-affinity interaction (K, = 900 PM). The high-affinity K, value cannot be estimated more precisely because of the low level of the signal in this range. Similar plots and K, values are observed for CTP and dATP (data not shown). In the presence of AMPPCP at 5 mM, a single apparent K, value for ATP is observed at about 26 t.rM (Figure 5). This probably reflects saturation of the low-affinity site by AMPPCP where it can substitute for ATP, combined with competitive inhibition at the high-affinity site; the V,, remains unchanged. Further Opening of the Duplex in the Prepriming Complex Following incubation at a high temperature with dnaB and dnaC proteins, a prepriming complex that also includes these proteins at oriC can be isolated. Subsequent stages of replication can then occur at 16%. Prepriming complexes and possible subassemblies were probed with Pl

nuclease at 16% (Table 4) to assess which components are required to stabilize the Pl-sensitive open complex at a low temperature. Sensitivity to Pl nuclease required dnaA, dnaB, and dnaC proteins and an incubation at elevated temperature, and was stimulated by HU protein (Table 4). This agrees with previous studies of prepriming complex formation using replication (van der Ende et al., 1985) or extensive unwinding (Baker et al., 1986) as assays. Along with the template becoming available for priming and replication, this sensitivity to Pl nuclease suggests that part of the duplex DNA in the prepriming complex has been melted into single strands. Mapping of the Pl cleavage sites in the prepriming complex was carried out as for the open complex, and the sites were compared with those in the open complex. The pattern of cuts is different in the two types of complex, particularly in the upper strand (Figures 3C and 3D). The greater stability of the prepriming complex at the lower temperature, as well as the different pattern of cuts, implies DNA-protein interactions in the prepriming complex distinct from those in the open complex. The nucleotide requirement for forming the stable prepriming complex differed from that described for the initial actions of dnaA protein. Whereas ATP dATF, and CTP are effective for formation of the open complex, the prepriming complex is uniquely dependent on ATP (Table 3). Thus, at least one interaction at this step requires ATP in a manner distinct from that observed in duplex opening by dnaA protein. The Prepriming Complex Blocks the Bglll Nuclease Cleavages in oriC Electron microscopic measurements (Funnell et al., 1987) have shown the prepriming complex to be larger and less symmetrical than the initial dnaA-oriC complex and to cover the DNA at the left end of oriC in the region of the Bglll restriction sites. The plasmid pCM959 (Buhk and Messer, 1983) contains four Bglll recognition sites, two of which lie at the left end of oriC, overlapping the leftmost and middle 13-mers; the other two are 500 and 1800 bp distant from OX (Figure 6). Bglll nuclease can bind its recognition sequence at a low Mg*+ level (an optimum condition for maintaining the prepriming complex), but re-

Cell 740

Bglll
C 38 38 38 22

25

dnaA dnaB dnaC

000

btc I..ML--.-.

--a+C

15

20

25

30 (C)

35

40

-+I,

TEMPERATURE

Figure 7. Temperature Profiles of Formation of Open and Prepriming Complexes on Supercoiled pBSoriC+ DNA As Monitored by Pl Nuclease Sensitivity Open complexes were formed and probed for 5 set with 1.2 U tions lacking dnaA protein (240 and the difference was plotted were formed at the temperatures 16C and probed with 1.2 U of tein-independent values were plotted (closed circles). in 2 min at the temperatures indicated of PI nuclease. Values for control reacng) wereobtained at each temperature, (open circles). Prepriming complexes indicated for 20 min, then cooled to PI nuclease for 5 sec. The dnaA prosubtracted, and the difference was

Figure 6. Prepriming Nuclease in oriC

Complex

Formation

Abolishes

Cutting

by Bglll

Complexes were formed at 36OC or 22C on 150 fmol of pCM959 DNA as described in Experimental Procedures. Reactions were cooled to 22OC, and 25 U of Bglll was added and incubated for 5 min at 22oC. The magnesium concentration was then raised from 0.3 mM to 10 mM to allow cleavage for 5 set before being stopped with EDTA. Samples were extracted with phenol and end-labeled, using DNA polymerase I large fragment, before electrophoresis through a 1.2% agarose gel. Fragments marked by arrows on the left can be generated only by cutting in oriC; those marked on the right can be generated without cutting in OK. Lowercase letters refer to the fragments indicated in the map of pCM959 (above).

quires a high magnesium concentration (e.g., 10 mM) for efficient cleavage. Protein-DNA complexes were allowed to form for 50 min at a low Mg*+ level either at 38C or 22%, before addition of Bglll nuclease. Incubation was continued at 22% at the low Mg*+ level to enable Bglll to bind (but not cleave) its recognition sequences. Then Mg*+ was added to 10 mM to initiate cleavage by Bglfl during a 5 set period. Analysis of the partial restriction products (Figure 8) shows that significant protection of the two Bglll sites in oriC was observed only in the presence of HU, dnaA, dnaB, and dnaC proteins and required incubation at an elevated temperature, the condition needed for forming the prepriming complex. Protection of the Bglll sites in oriC after cooling at 22C is further evidence of the stability of the DNA-protein interactions in the prepriming complex. At 38% the open complex does provide limited protection (9fold) against Bglll cleavage (T A. Baker, D. Bram-

hill, and A. Kornberg, unpublished data). Two factors may explain the low level of protection: First, the affinity of dnaA protein for the 19mers may be similar to that of Bglll for its recognition sequence. Thus, Bglll might displace the equilibrium, reducing open complex formation. Second, although numerous Pl cuts map in the leftmost 19mer, the majority lie in the middle and rightmost 13-mers. Since one 13-mer is sufficient for open complex formation (see below), then it is possible that most open complexes are still double-stranded in the leftmost 13-mer and are therefore Bglll-sensitive. Temperature Profiles for Partial and Full Opening of the Duplex at 0riC Formation of both the open and prepriming complexes by dnaA protein requires an elevated temperature (van der Ende et al., 1985). The extents of open and prepriming complex formation were measured at different temperatures. To determine the minimum temperature for formation of the open complex, probing with Pl nuclease was carried out at the temperature at which the complex was formed, inasmuch as these complexes are cold-sensitive. dnaA protein was omitted from reactions at each temperature to control for the influence of temperature on Pl activity. Open complex formation was clearly evident at 23% (Figure 7). Prepriming complexes were formed at a given temperature, and then probed with Pl at 18%. Their appearance required a temperature above 28%. This direct measurement agrees well with observations using replication (van der Ende et al., 1985) or dnaB helicase action (Baker et al., 1986) as assays of prepriming complex formation. Thus, open complex formation is observed at a

;;;A-Catalyzed

Duplex

Opening

I/i,,,/ III

ArcI

ORI PHENOTYPE +

Figure

8. Origin

Regions

of Wild Type oriC and Mutant

Derivatives

Arrows represent

15mers

(see Figure 1). and stippled

boxes represent

9-mers. A4 bp insertion at the Hindlll site is indicated by a light stippled box; deletions are indicated by gaps. Restriction sites used in the constructions are indicated (for details see Experimental Procedures). The figure is not drawn to scale. The ori phenotype of each plasmid is indicated. pBSoriC+ DNA transformed a PO/A- host at least WOO-fold more efficiently than did any of the oriC mutant plasmids (no transformants recovered). All plasmids transformed a PO/A+ host with a similar efficiency (about 2 x lo4 per pg of DNA).

tant plasmids failed to function in the PO/A- host, but could transform the isogenic PO/A+ strain. These observations are consistent with the oriC sequences extraordinary evolutionary conservation (Zyskind et al., 1983) and its sensitivity to mutational changes (Oka et al., 1980, 1982, 1984). Sequential stages of initiation in vitro were then examined to determine the basis for these mutational losses of function. Formation of the Open Complex Requires One Intact Id-mer The mutated oriC plasmids were tested for their ability to form open complexes with dnaA protein. Pl nuclease was used at 38% to monitor complex formation. Deletion of the left 13-mer (oriCA13L) or two 13-mers (ofiCA13LM) has no detectable effect either on the level of induced Pl
sensitivity or on the amount of dnaA protein required (Figure

of the subsequent prepriming complex formation. The optimal Mg2+ level of 8 mM was used for forming the open complex, in contrast to the Mg*+ level of 0.3 mM used for forming the prepriming complex. Because a higher Mg2+ level is expected to raise the temperature for open complex formation, the temperature difference (between open and prepriming complex formation), if measured at the same Mg2+ level, might be even greater than 4%. These findings on the influence of temperature are in keeping with the behavior of the mutated replication origins (see below). Only one 13-mer (theoretically, only a single base pair) need be opened to generate a Plsensitive site, and this can occur at 23%. However, to allow dnaB to enter and generate a stable prepriming complex, all 13-mers must be opened and kept open. Since each opened 13-mer eliminates more than one superhelical turn from the plasmid, successive openings are energetically less favorable-hence the requirement for a higher temperature. Deletion Analyses To investigate the role of the 13-mers in more detail, deletions in oriC were constructed. The properties of each mutated origin were then examined in vivo and in vitro. In Vivo Function of oriC Requires All Three Id-mers and Four 9-mers Each of five mutations in the 0% sequence, deletion derivatives of oriC (lacking one, two, or all three 13-mers, or the rightmost 9-mer), and an insertion between the two righthand 9-mers (Figure 8), completely destroyed the capacity of an oriC plasmid to transform a PO/A strain (data not shown). The host, lacking pa/A function, cannot support the pBR322-derived vector origin and must therefore depend on oriC function to maintain the plasmid. The mu-

temperature than that

at least

4%

lower

(at the transition

midpoint)

9). However, deletion of all three 13-mers (oriCAl3LMR) abolishes duplex opening. Thus, the dnaA protein appears to require at least one 13-mer to generate a singlestranded region; mere AT-richness, supplied by the contiguous DNA to the left of ofiC in ofiCAl3LMR, does not suffice for duplex opening. Mutations at the right end of oriC have little effect on the reaction. Sites of P7 Sensitivity in the Mutated oriC Sequences Lie in Intact Id-mers Mapping of the PI-sensitive sites in the mutant origins confirmed their location in the 13-mers that remained intact. The open complex was formed with each DNA, linearized at 38% by Pl nuclease, and finally analyzed in native gels and sequencing gels (Figure 10). In the native gel (not shown), virtually all (>95% as determined by densitometric scanning) of the Pl cuts are seen in the 13-mer re-

60

120 160 240 dnaA PROTEIN (ng)

Figure 9. Open Complex Formation by Pl Nuclease Sensitivity

on oriC Mutant

DNAs,

Monitored

Reactions were carried out as described in Experimental Procedures, and contained 150 fmol of supercoiled DNA (see Figure 8) and dnaA protein, 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 being quenched with 40 pl of 25 mM EDTA, 1% SDS. The proportion of linearized molecules was determined by densitometric scanning of photographic negatives of 0.7% agarose gels stained with ethidium bromide. Values with oriCAl3LMR are identical to those with oriC completely deleted.

Cell

750

DNA

60 -

L L
13.mers M 0 60 120 160 240 dnaA PROTEIN (ng)

*a

Figure 11. Prepriming Complex Formation Monitored by Pl Nuclease Sensitivity

with OK

Mutant

DNAs,

Prepriming complexes were formed at 38OC for 30 min with supercoiled DNA (150 fmol) as indicated. Reactions were chilled to 18OC and probed for 5 set with 1.2 U of PI nuclease before being quenched with 40 ul of 25 mM EDTA, 1% SDS. The proportion of linear molecules was determined from densitometric scanning of photographic negatives of gels stained with ethidium bromide. The dnaA-independent background values (4%-10%) have been subtracted in each case. Values with oriCA13LM are identical to those with oriCAl3LMR and with oriC completely deleted.

Figure 10. Nuclease the oriC Mutants

Cleavage

Sites in Open Complexes

Formed

with

Open complexes were formed with DNAs (150 fmol) and dnaA protein (240 ng) as indicated and probed with Pl nuclease. Linearized DNAs were dephosphorylated and 5end-labeled using T4 polynucleotide kinase. Incubation with Hindlll generated fragments within oriC of about 200 bp (see Figure 8) that were resolved on a 8% polyacrylamide sequencing gel (80 cm x 0.2 mm). The arrows indicate the positions of the 18mers in the wild type sequence, deduced from sequencing reactions and Bglll-Hind81 oriC fragments run in the same gel.

gion of oriC. The sequencing gel shows that in the case of wild type oriC or oriCA13L, the cuts lie predominantly in the middle 13-mer. When 4 bp more (the first 4 bp of 19mer M) are deleted from oriCA13L to give oriCA13LM, the major sites cut by Pl lie in the only intact 19mer, the rightmost, and not in the remaining 9 bases of the middle 19mer. These results strongly support the idea that dnaA protein specifically recognizes the 13-mer sequence or some portion of it. Formation of the Prepriming Complex Requires A// Three l&mers Mutational changes in oriC have a more profound effect on formation of the prepriming complex than on formation of the open complex. Efficient formation of the prepriming complex requires all three 13-mers (Figure 11). Even the loss of only one (oriCA13L) results in a very low level of

dnaA-dependent Pl sensitivity at low temperature. These results are consistent with the temperature profiles of open and prepriming complex formation (Figure 7). Neither deletion of the rightmost 9-mer repeat (oriCA9R) nor insertion of 4 bp between the two rightmost 9-mers (ofiCV9R) appears to alter the ability to support formation of the prepriming complex. Still, as noted earlier (Figure 8) these mutants are inactive in vivo. Replication In Vitro Parallels Prepriming Complex Formation Two reconstituted enzyme systems were examined for their capacity to replicate the mutant plasmids. In the solo primase system (van der Ende et al., 1985) a low level of protein HU is present (similar to the level of HU in the Pl assays) to stimulate the reaction and RNA polymerase is absent. In the other system, higher levels of HU protein partially coat the plasmid and RNA polymerase transcription is required to activate the origin; RNAase H is also needed to maintain oriC specificity (Kaguni and Kornberg, 1984; van der Ende et al., 1985; Ogawa et al., 1985). Participation of RNA polymerase in the initiation of replication is also indicated by earlier findings (Zyskind et al., 1977; Fuller et al., 1981). In response to mutational changes in oriC, the solo primase system (Figure 12A) shows a replication pattern similar to that observed for formation of the prepriming complex. Deletion of the left 19mer greatly reduces replication activity; further deletion of 4 bp of the middle 13-mer abolishes replication. On the other hand, mutations at the right end of oriC cause little reduction. By contrast, repli-

ch:A-Catalyzed

Duplex

Opening

(A) Solo Primase

30

60

90

120 dnaA PROTEIN (ng)

Figure

12. In Vitro Replication

of Mutant

oriC Plasmios for 20 min at 30%. (A) Solo primase fraction II system. (See Experimental

Reactions (25 pl) containing supercoiled plasmid DNA (75 fmol) and dnaA protein as indicated were incubated reconstituted enzyme system. (Et) RNA polymerase-dependent reconstituted enzyme system. (C) dnaA mutant Procedures.)

cation in the RNA polymerase-dependent system (Figure 126) resembles the behavior of the mutants in vivo. Deletion of even one 13-mer abolishes the capacity of the plasmid to serve as a template. Even mutations at the right end of 0% enfeeble the plasmid in this system. Presumably, the higher levels of HU protein that lead to the requirement for RNA polymerase activation also contribute to a more stringent requirement for the intact oriC sequence. At the higher HU concentrations, more dnaA protein is also required to activate the origin. In a crude enzyme fraction (Fuller and Kornberg, 1983), much as in the RNA polymerase-dependent reconstituted system, replication of mutated templates (oriCAQR, oriCVQR, and ofiCA13L) is greatly affected (Figure 12C). These results, so similar to those observed in vivo, suggest that both the crude enzyme fraction and the RNA polymerase-dependent reconstituted system closely reflect the cellular conditions. Discussion By analyzing deletions in oriC and using Pl nuclease as a probe for single-stranded DNA, we have demonstrated that the duplex is opened by the action of dnaA protein at oriC and is maintained in an even more stable open form by the prepriming complex. Earlier mutational studies assessed origin function only in vivo (Oka et al., 1982, 1984) and were limited essentially to an all-or-none result. The parallel in vitro studies presented here allow a much more refined analysis of altered origins. Deletions in the 19mer region were readily obtained by exploiting the 13-merspecific cleavage of the open complex by Pl nuclease, and the sites of cleavage by certain restriction nucleases. The stage at which each mutation blocks initiation has been determined, and the residual levels of activity at this and subsequent steps have been measured. The possibility remains that the DNA does not become single-stranded, but takes on some other conformation that is Pl-sensitive. However, bending or kinking of the DNA is inconsistent with the observed dnaAdependent Pl cutting of opposite strands within one or two bases.

Bending could enhance attack only on one strand at any point. That the structure is not Z-DNA is shown by the cold sensitivity of the open complex. The simplest explanation is that the 13-mer DNA is indeed single-stranded in the open complex. This is consistent with the documented specificity of Pl nuclease (Kowalski, 1984) the susceptibility of both strands to Pl attack, the requirement for high temperature and sensitivity to cold, and also the availability of the template for subsequent dnaB helicase action. The 13-mer Sequence At the left end of oriC, three 13-mers in tandem have been identified, coinciding with the site for duplex opening by the dnaA protein in the initiation of replication. This suggests that dnaA protein specifically recognizes these 13-mer sequences. Data supporting this idea include the following: First, two such 13-mers are located close to the dnaA box in the origin of a plasmid (pSC101) also dependent on dnaA protein. Second, the 13-mer sequence is almost as highly conserved among oriC sequences in distantly related Enterobacteriaceae as are the dnaA boxes, the only deviations being in the sixth and ninth positions, the two nonspecific positions in the consensus. Third, deletion of either the leftmost or part of the middle 13-mer inactivates origin function in vivo, whereas an insertion of 4 bp between them is tolerated (Oka et al., 1980). Fourth, deletion mutants of oriC lacking the left and middle 13mers can still form an open complex that is sensitive to Pl nuclease, specifically in the remaining (right) 13-mer, while removal of all three 13-mers abolishes duplex opening. Fifth, many other prokaryotic origins possess equivalent tandem repeats in their AT-rich regions; these origins include those of h, @80, @82, Pl, F, Rl, R8K, RK2, and P4, as well as the putative Bacillus subtilis oriC (Bramhill and Kornberg, unpublished data). A Model for OK Initiation The insights provided by probing with the single-strandspecific Pl nuclease enable us to refine our model of the early initiation events at oriC. We propose the following

Cell 752

Figure

13. A Scheme

for oriC Initiation negadnaA work 1984;

Details are given In the text. The 1 to 1% tive turns of oriC DNA wrapped about the protein core is suggested from earlier (Fuller and Kornberg, 1983; Fuller et al., Funnell et al., 1987).

SUPERCOILEO TEMPLATE AND REPLICATION COMPLEX

stages (when HU protein at a stimulatory level coats about 3% of the plasmid) (Figure 13): First, dnaA protein binds to its 9 bp boxes in OK. Second, an initial dnaA-oriC complex is formed, incorporating 20-40 monomers of dnaA in a central core around which oriC DNA is wrapped. Third, stepwise duplex opening of the 13-mers by dnaA protein occurs at an elevated temperature to form an open complex. Fourth, the dnaB and dnaC proteins associate (probably as a B-C complex) with the open complex by protein-protein interactions. Fifth, the dnaB helicase enters to produce a prepriming complex; the helicase probably recognizes the single-stranded DNA structure, a potential fork, rather than the sequence of the 13-mers from which it probably displaces dnaA protein. Further dnaB helicase action, upon addition of SSB and gyrase, unwinds the template and exposes it to action by primase. Guided by dnaB, primase may synthesize RNA primers that DNA polymerase Ill holoenzyme can extend. Open Complex Formation Open complex formation requires only the dnaA protein, as indicated by the copurification of the duplex-opening activity with the replication-initiation activity of the nearly homogeneous dnaA protein, and the persistence of the Pl nuclease cutting pattern in the 13-mers in the presence of only the dnaA protein (omitting IHF and HU proteins). Retention of only one of the three 13-mers suffices for the formation of a Pl-sensitive open complex at 38%. Although the deletions oriCAl3L and oriCAl3LM result in equally AT-rich contiguous DNA sequences replacing the deleted DNA, mapping reveals that the Pl cuts lie almost exclusively in the remaining 13-mers and not in these adjacent AT-rich sequences. These results suggest that dnaA protein specifically recognizes all or part of the 19mer sequence. Since each 13-mer includes a dam methylation sequence, specific recognition by dnaA protein of one or both of the modified adenines in the 13-mer could account for the observed effects of dam methylation in vivo and in vitro (Messer et al., 1985; Smith et al., 1985). A simple experimental approach to defining the precise sequence requirement for dnaA protein recognition would be to analyze point mutations in a single 13-mer for open complex formation by the dnaA-dependent Pl cutting assay.

The site-specific duplex opening activity of dnaA protein implies that dnaA may be able to recognize two distinct DNA sequences. One is the 9 bp dnaA box, as a duplex, and the other the 19mer as either double- or singlestranded DNA. A similar dual sequence specificity has been found in the actions of phage lambda site-specific int recombinase (Ross and Landy, 1982, 1983). The int enzyme first binds three specific arm sites in a supercoiled donor DNA substrate, organizes the DNA around a central core of protein (Richet et al., 1986), and then recognizes in both the donor and acceptor molecules a second junction sequence that serves as the site for strand exchange. Similarities can also be seen between the 13-mers and promoters for RNA polymerase, both at the structural and functional levels. Like a transcriptional promoter, the 13-mers in oriC serve as the entry site for an enzyme to invade a duplex DNA region. Both types of entry sequence are AT-rich, thereby facilitating strand separation. Sharp temperature transitions are observed in the formation of open complexes by RNA polymerase and by dnaA protein. Each 13-mer opened will lead to the loss of slightly more than one superhelical turn per template. Thus, successive opening events will require more energy. A consideration of the physical constraints imposed by the dnaA protein complex binding tightly to the adjacent portion of oriC suggests that the opening events may be sequential, starting with the rightmost 13-mer, nearest the 9 bp dnaA box (Figure 14). Such a stepwise mechanism avoids several problems of a one-step mechanism by requiring recognition of only a short DNA sequence such as can be achieved by a helix-turn-helix or an equivalent protein domain, and lower activation energies for individual opening events. This stepwise mechanism also facilitates opening up just over one helical turn at a time and realigning the DNA for the next opening step. The nucleotide requirements for dnaA-dependent open complex formation are rather complicated. Still, our observations might be explained by a single ATP site per dnaA molecule. Of the 20 or more dnaA subunits in the open complex, some may bind the 9-mers and some the Wmers, some interact with the dnaBC complex, while yet others may only contact adjacent monomers. Such varied inter-

dn;A-Catalyzed

Duplex

Opening

may also provide a key to regulation: production of inactive ADP-dnaA protein within the complex coupled to dnaB helicase entry would insure a single initiation per origin. Prepriming Complex Formation The Pl-sensitive structure formed by dnaA protein at 38% in the presence of ATP is unstable at 16%. To produce a complex that retains a Pl-sensitive configuration at 16%, dnaB and dnaC proteins are both required during the 38% incubation to form a prepriming complex. Thus, dnaB helicase or dnaC, rather than dnaA protein, appears to be responsible for maintaining the single-stranded region in the prepriming complex. Such a role for dnaB protein can explain the difference in the Pl-sensitive sites between the dnaA open complex and the prepriming complex. Protection of the prepriming complex from Bglll restriction nuclease cleavage also supports direct dnaB or dnaC involvement and demonstrates that the prepriming complex includes the 13-mer region. This is consistent with the earlier findings by electron microscopy (Funnell et al., 1987) that approximately 50 bp of the left end of oriC is included in the prepriming complex, but not in the initial complex with dnaA protein alone. Efficient formation of a stable prepriming complex requies all three 13-mers and an elevated temperature. These observations suggest that while the sensitivity to Pl of the open complex requires that only one 18mer be recognized by dnaA protein, efficient entry of dnaB helicase depends on having all three 13-mers opened. This proposal is reasonable in view of the larg$ size of the hexameric dnaB (300 kd; Stpkes radius, 58 A) or dnaBC (480 kd; Stokes radius, 64 A) complexes (Kobori and Kornberg, 1982). The higher temperature required for forming the prepriming complex is also consistent with this model. The formation of a prepriming complex exhibits a nucleotide requirement in addition to that for open complex formation. Only ATP is able to support prepriming complex formation, whereas CTP and dATP are both effective for duplex opening. If there is in fact only a single site for ATP (shared by CTP and dATP) per dnaA monomer, then this absolute requirement for ATP to form a prepriming complex most likely reflects an interaction with dnaB or dnaC proteins, as in the formation of a dnaBdnaC complex (Wickner and Hurwitz, 1975; Kobori and Kornberg, 1982). Many other origins have an organization similar to oriC, including those of lambdoid phages, certain broad hostrange plasmids, and even SV40. The SV40 T antigen leaves a footprint on portions of the SV40 minimal origin (Tjian, 1978), but not on an essential AT-rich region (Stillman et al., 1985). The T antigen also possesses a helicase activity (Stahl et al., 1986; Dean et al., 1987). The model based on the actions of dnaA protein in initiating oriC may prove to be applicable to initiation of a wide range of origins, prokaryotic and eukaryotic.
Experimental Procedures

) dnaA A lllll ) dnaA ,, II

Figure 14. Proposed tein and Subsequent

Sequential Opening of the 13-mers Entry of the dnaB-dnaC Complex by arrows, the leftmost 9-mer

by dnaA Proby a stippled

The 13-mers are indicated box (cf. Figure 8).

actions could result in significant differences in affinities for ATP and its functions. Three distinct requirements for ATP in formation of an open complex are implied by three apparent K, values: The first is a very tight binding of ATP and also of ADP, CTP, dATP, and ATPyS, with a KD of 30 nM (Sekimizu et al., 1987). ATP activates dnaA protein for replication, while ADP blocks strand opening, even in the presence of 5 mM ATP; release of the bound nucleotide is very slow (tl,Z of 30 min). Hydrolysis of the tightly bound ATP to ADP is equally slow. Inasmuch as tightly bound ATPyS can also activate dnaA protein for strand opening, in the presence of 5 mM ATP, an allosteric role for this interaction with ATP seems likely. A second requirement for ATP has a K, near 1 PM. Although CTP and dATP substitute for ATP, some properties distinguish this interaction from the highaffinity interaction. Analogs such as ATPyS, AMPPNP, and AMPPCP are inactive but appear to inhibit competitively. The need for a hydrolyzable nucleotide implies that in this case ATP may provide energy either for strand opening or for helicase entry. A third requirement for ATP has a low affinity, with a K,,, around 1 mM. As in the other two cases above, dATP and CTP can substitute for ATP Analogs such as ATPyS and AMPPCP are also effective here, suggesting a second allosteric interaction of dnaA protein with ATP That the same set of nucleoside triphosphates (ATP, CTP, dATP) is effective in each case, and that no more than a single ATP molecule is bound per dnaA subunit in nitrocellulose filter binding (Sekimizu et al., 1987), argues in support of a single site. This model also reconciles the requirement for a hydrolyzable nucleotide for open complex formation (K, < 1 PM) with the very low level of ATP hydrolysis observed during prepriming complex formation (Sekimizu et al., 1987), since it explains why hydrolysis of ATP might occur on only one or a few crucial subunits of dnaA in the complex. Such an essential hydrolysis of ATP

Reagents ATP. deoxyribonucleoside

triphosphates,

and HEPES

were from Phar-

Cell 754

macia; GTR CTP UTP and ADP were from Sigma; ATPTS. AMPPNP, and AMPPCP were from Boehringer; [Y-~~P]ATP (7000 Cilmmol) was from New England Nuclear; [a-32P]dATP (3000 Cilmmol) was from Amersham. Enzymes and Proteins Samples of purified E. coli IHF were gifts from H. Nash (National Institutes of Health) and N. Cozzarelli (University of California, Berkeley); both preparations are essentially free of contaminating HU protein as judged by SDS-polyacrylamide gel analysis. Purified replication proteins were prepared as described by Kaguni and Kornberg (1984). The dnaA protein was purified by the method of Fuller and Kornberg (1983) to a specific activity of 1.0 x 106 Ulmg and a purity greater than 90% as judged by SDS-polyacrylamide gel electrophoresis. Restriction enzymes, T4 polynucleotide kinase. T4 DNA ligase, and Sl nuclease were from New England Biolabs; calf intestinal phosphatase was from Boehringer; Pl nuclease was from Pharmacia. Bacterial Strains E. coli strains MM383 @o/A72 lacZ53 rpsLl51 thyA rba-5 deoC2 IN(rmD-rrnE)l) and MM384 (isogenic pa/A+ control) (Monk and Kinross, 1972) were used for testing oriC function in vivo. Strain DHl (recA1 endA hsdFfl7gyrA96supE44 fbi-1) (Hanahan, 1983) was used for mutant constructions. Construction of Mutant oriC Plasmids Plasmid pBSoriC contains a 678 bp Hincll-Pstl fragment spanning oriC (-189 or +489) cloned into the pBluescript vector (Stratagene, Inc., San Diego) (T Baker and A. Kornberg. unpublished data). The Bglll sites at the left end of oriC allow the specific deletion of the leftmost 13-mer (pBSoriCA13L) or, following Sl nuclease trimming, the left and 4 bp of the middle 13-mer (pBSoriCA13LM). More extensive deletions of the left end of oriC were constructed by exploiting the Pl nuclease sensitivity induced by dnaA protein. Pl-linearized pBSoriC DNA molecules were recovered from a 0.7% low melting point agarose gel (Seaplaque), restricted with Bglll, and isolated from a second low melting point gel run as almost full-length linear species free from shorter DNAs. Religation generated a series of deletions. The shortest deletion, designated pBSoriCA13LMR lacked the leftmost 43 bp of oriC. The Hindlll-Accl fragment was deleted to produce pBSoriCASR, and 4 bp insertion at the Hindlll site (pBSoriCV9R) was produced by filling in the Hindlll sticky ends by DNA polymerase I. In each case, religation was performed at 10 uglml DNA to favor recircularization. pCM959 (Meijer et al., 1979) a gift from M. Meijer (Amsterdam), is a 4012 bp minichromosome containing oriC (-677 to +3335). Plasmid DNAs were prepared as described by Ogawa et al. (1985). Pl Linearization of Open Complexes The standard reaction (50 pl) contained 40 mM HEPES-KOH (pH 7.6) 8 mM magnesium acetate, 30% (vol/vol) glycerol, 320 pglml BSA, 150 fmol of supercoiled plasmid DNA, 67 ng of HU protein, 240 ng of dnaA protein, and 5 mM ATP Following incubation at 38% for 2 min, 1.2 U of Pl nuclease was added in 3 ul of 30 mM potassium acetate (pH 4.8). Pl cleavage was stopped after 5 set by the addition of 40 ul of 25 mM EDTA. 1% SDS. For quantitation of the fraction of linear molecules, a portion of the reaction was electrophoresed through a 0.7% agarose gel in 100 mM Tris-borate (pH 8.3) 1 mM EDTA (TBE) at 6 V/cm, stained with ethidium bromide, and photographed using Polaroid film. Densitometric scanning of the negative was used to determine the proportion of linear molecules. Pl Linearization of Preprimlng Complexes Reactions, in 50 ul, were similar to the open complex reactions except dnaB (120 ng) and dnaC (40 ng) were included, and the free magnesium concentration was adjusted to 0.3 mM by addition of EDTA to chelate excess magnesium contributed by theenzyme buffers. Incubation at 38% was for 30 min, after which reactions were chilled to 16% and Pl nuclease (1.2 U) was added. After 5 or 10 sec. digestion was stopped by the addition of EDTA and SDS as for open complexes. Quantitation was also by identical methods. Mapping Pl Cut Sites Linearized DNAs were extracted with phenol-chloroform, precipitated with ethanol, and washed with 70% ethanol. Following dephosphoryla-

tion and 5 end-labeling with [y-s*P]ATP using T4 polynucleotide kinase, DNAs were restricted with appropriate enzymes and prepared for electrophoresis. To obtain nucleotide-level resolution, urea-polyacrylamide sequencing gels (60 cm x 0.2 mm) were run on an LKB 2010 Macrophor apparatus at 65C 2300 V for 2-4 hr in TBE buffer. Maxam-Gilbert sequencing reactions, DNAase I digestion ladders, and Bglll restriction fragments recutwith the appropriate enzyme were used as standards to locate the positions of the PI cuts. In Vitro Replication Assays The following methods have all been described previously: RNA polymerase-dependent reconstituted replication assays (Ogawa et al., 1985); solo primase reconstituted replication assays (van der Ende et al., 1985); and dnaA mutant fraction II complementation assays (Fuller and Kornberg, 1983). Acknowledgments We are grateful to L. Bertsch for careful reading of the manuscript, and to D. Brutlag for advice and assistance in computer searches. This research was supported by grants from the National Institutes of Health and the National Science Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received October 13, 1987; revised December 31, 1987.

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Opening

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Zyskind, J. W., Deen, L. T., and Smith, D. (1977). Temporal sequence of events during the initiation process in E. co/i DNA replication: roles of the dnaA and dnaC gene products and RNA polymerase. J. Bacteriol. 129, 1466-1475. Zyskind, J. W., Cleary, J. M.. Brusilow, W. S. A., Harding, N. E., and Smith, D. W. (1983). Chromosomal replication origin from the marine bacterium Vibrio barveyi functions in Escbericbia co/i: oriC consensus sequence. Proc. Natl. Acad. Sci. USA 80, 1164-1168.

Sekimizu, K., Bramhill, D., and Kornberg. A. (1987). ATP activates dneA protein in initiating replication of plasmids bearing the origin of the E. coli chromosome. Cell 50, 259-265.