complete enzymatic replication of plasmids …0 1986 by the american society of biological chemists,...

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1986 by The American Society of Biological Chemists, Inc

Vol. 261, No. 12, Issue of April 25, pp. 5616-5624 1986 Printed in G.S.A.

Complete Enzymatic Replication of Plasmids Containing the Origin of the Escherichia coli Chromosome*

(Received for publication, November 13, 1985)

Barbara E. FunnellS, Tania A. Baker, and Arthur Kornberg From the Department of Biochemistry, Stanford Uniuersity School of Medicine, Stanford, California 94305

During enzymatic replication of plasmids containing the origin of the Escherichia coli chromosome, oriC, formation of an active initiation complex consisting of dnaA, dnaB, dnaC, and HU proteins, requires a supercoiled DNA template. Relaxed covalently closed plasmids are active only if supercoiled by gyrase prior to initiation; nicked and linear DNAs are inactive. Semi- conservative replication proceeds via 0 structures as intermediates. Daughter molecules include nicked monomers and catenated pairs. Elongation is rapid, but late replicative intermediates accumulate because the final elongation and termination steps are slow. Production of covalently closed circular daughter DNA molecules requires removal of ribonucleotide residues (primers) by DNA polymerase I, assisted by ribonu- clease H, gap filling, and ligation of nascent strands by ligase. Reconstitution of a complete cycle of oriC plasmid replication, beginning and ending with supercoiled molecules, has been achieved with purified proteins.

Biochemical studies of replication directed by the Esche- richia coli chromosomal origin (oriC) became possible after the development of a soluble enzyme system capable of spe- cifically replicating oriC-containing plasmids (1). Analysis of DNA intermediates and products was complicated by a mas- sive catenation of plasmid DNA very early in the reaction. The catenation was catalyzed by topoisomerase I and exonuclease 111 in the presence of hydrophilic polymers which were required for DNA synthesis with crude enzyme systems (2). Replicative intermediates could be isolated and analyzed only after linearization by restriction enzymes (1, 3). Thus, infor- mation regarding the structure and topology of intact DNA intermediates and products could not be obtained.

Recently, oriC-specific replication has been partially reconstituted with purified proteins in the absence of hydrophilic polymers (4-6), and the catenation problem avoided. Repli- cation has been separated into a series of initiation and elongation stages (5, 6). We have now examined the requirement for supercoiling of oriC plasmids during initiation, in view of the observations that closed circular DNA is negatively supercoiled in uiuo, and DNA topology is known to strongly influence both replication and transcription (for review, see Ref. 7). We have also analyzed the structure of the DNA products throughout the replication cycle, and de-

* This work 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 “aduertise- ment” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Supported by a fellowship from the Medical Research Council of Canada.

termined which enzymes are required to form supercoiled daughter molecules.

EXPERIMENTAL PROCEDURES

Reagents-Sources were as follows: ribonucleoside triphosphates, phosphocreatine, and Tricine; Sigma; [ W ~ ~ P I ~ T T P and [a-”P] dATP, Amersham Corp; [3H]dCTP, New England Nuclear; deoxyri- bonucleoside triphosphates, bromodeoxyuridine triphosphate (BrdUTP) and NAD, P-L Biochemicals; Hepes, Calbiochem-Behring; bovine serum albumin (BSA), Pentex; Bio-Gel A-5m, Bio-Rad.

Enzymes-Highly purified replication proteins were prepared as previously described (4). DNA polymerase I (pol I) was purified essentially as described (8). E. coli DNA ligase was a gift from I. R. Lehman (this department). Pancreatic DNase I was from Worthing- ton, and restriction enzymes were from New England Biolabs.

Plasmids-pCM959 (9), a gift from M. Meijer (University of Am- sterdam, The Netherlands), is a minichromosome (4012 bp) consisting solely of E. coli DNA encompassing oriC (bp -677 to i-3335). M13mpRE85 (7866 bp) was a gift from D. W. Smith (University of California, San Diego). It includes a 637”bp E. coli oriC fragment (bp -189 to +448) cloned into the PsB site of M13mp8 (10). M13oriC5BL2 (6653 bp) contains a 329-bp oriC fragment (bp -41 to +288) in M13AE101, an M13 derivative lacking the complementary strand origin. It was constructed by inverting the oriC fragment with respect to M13, of M13oriC2LB5, which is described elsewhere (4). The DNAs were prepared and purified as described (5).

Preparation of Different Plasmid Topological Forms-Relaxed plasmid was prepared by treatment with E. coli topoisomerase I: 10 pg of supercoiled plasmid DNA was incubated with 0.4 pg of topoisomerase I in 20 mM Hepes-KOH (pH 8), 20 mM KCl, 5 mM MgC12, 100 pg of BSA/ml, and 5% glycerol (volume = 100 pl) for 40 min at 30 “C (2). The reaction was stopped by the addition of SDS to 0.5% and NazEDTA to 10 mM, extracted twice with phenol/CHCb (1:1), precipitated with ethanol, and resuspended in 20 ~1 of 10 mM Tris-HC1 (pH 7.6), 1 mM EDTA. Nicked plasmid was prepared as follows: 10 pg of DNA was incubated with 6 pg of DNase I in 50 mM Tris-HC1 (pH 8), 50 mM NaCl, 10 mM MgC12, and 0.35 mg of ethidium bromide/ ml for 15 min at 30 “C. The ethidium bromide was subsequently removed by extracting with water-saturated butanol before phenol extraction and DNA isolation as above. Linear M13mpRE85 was prepared by digestion with the single-site restriction enzyme Puul according to the directions of the supplier. Agarose gel electrophoresis confirmed that the correct topological forms were produced before subsequent assays were performed.

Reconstitution Assays for DNA Replication-The standard reaction

8 mM; phosphocreatine, 2 mM; ATP, 2 mM; GTP, CTP, and UTP, (25 p1) contained Hepes-KOH (pH 7.6), 40 mM; magnesium acetate,

500 PM each; dATP, dGTP, and dCTP, 100 p~ each; [a-3zP]dTTP, 100 PM, 100-400 cpm/pmol; template DNA, 200 ng (600 pmol of nucleotide); BSA, 0.4 mg/ml; creatine kinase, 500 ng, protein HU, 20 ng; gyrase A subunit, 300 ng; gyrase B subunit, 150 ng; RNA polymerase (as indicated), 475 ng; dnaA protein, 120 ng; single-strand DNA binding protein, 400 ng; dnaB protein, 60 ng; dnaC protein, 20 ng;

The abbreviationsused are: Tricine, N-tris-(hydroxymethy1)meth- ylglycine; Hepes, 4 - (2 - hydroxyethyl) - 1 - piperazineethanesulfonic acid; BrdUTP, bromodeoxyuridine triphosphate; SDS, sodium dode- cy1 sulfate; BSA, bovine serum albumin; pol I, DNA polymerase I; dNTP, deoxynucleoside triphosphate; bp, base pair(s); kb, kilobase pair(s).

5616

Products of oriC Replication 5617

primase, 10 ng; and DNA polymerase I11 (pol 111) holoenzyme, 70 ng. (The pol I11 fraction used was deficient in /3 subunit; 75 ng of purified p was also included.) The mixtures were assembled at 0 "C and incubated as described below. Total nucleotide incorporation was measured in a liquid scintillation counter after trichloroacetic acid precipitation onto Whatman GF/C glass-fiber filters.

The time, temperature, and order of addition of the components were varied in different assays. In general, the prepriming and priming stages were separated from elongation. The reaction mixture containing all components except dNTPs was incubated for 12 min at 35 "C, shifted to 16 "C for 2 min, mixed with dNTPs, incubated at 16 "C (or 30 " C ) for 6 or 10 min, and stopped by the addition of Na,EDTA to 10 mM and SDS to 0.25%.

To measure the replication of the different topological forms of template, the mixtures (a volume of 30 p1 in these reactions only) included all components and were incubated at 30 "C for the indicated times. To measure the requirement for supercoiling during prepriming, the reaction was staged as follows: the prepriming reaction (20 pl) contained Tricine-KOH (pH 7.6), 30 mM; glycerol, 20%; NaZEDTA, 0.5 mM; ATP, 5 mM; and BSA, phosphocreatine, dnaA protein, dnaB protein, dnaC protein, protein HU, and DNA as above. No magnesium was added; however, about 0.5 mM was contributed by the protein solutions. The mixture was incubated for 30 min at 38 "C and shifted to 24 "C, the remaining components except dNTPs were added and incubated for 4 min at 24 "C, and dNTPs were then added and DNA synthesis was measured after 20 min at 16 "C. When gyrase (but no other replication proteins) was added before prepriming, the DNA was incubated in the above buffers plus 8.5 mM magnesium acetate with gyrase A and B subunits for 10 min at 24 "C. The reaction was stopped with Na2EDTA (to 17 mM) and prepriming and subsequent stages were performed as above.

Replication with BrdUTP and CsCl Gradient Analysis of Products- Replication reactions (6-fold, 150 pl) contained pCM959 DNA and RNA polymerase in addition to the above components. BrdUTP was substituted for dTTP and [o(-32P]dATP for [cz-~'P]~TTP. After replication, the EDTA-treated reactions were incubated with RNase A (10 pg/ml) for 30 min at 30"C, stopped with SDS (to 0.2%), and deproteinized by gel filtration over a 5-ml Bio-Gel A-5m column in 20 mM Tris-HC1 (pH 8), 1 mM Na2EDTA, and 50 mM NaCl at 20°C. Void-volume fractions were determined by radioactivity and pooled.

The marker DNAs were 3H-labeled @X174 double-stranded forms. The light-light density marker, 3H replicative form, was a gift from R. Bryant (this department). Hybrid density DNA was synthesized in vitro from viral single-stranded circular DNA using purified replication proteins essentially as described previously ( l l ) , except BrdUTP (to 100 pM) and [3H]dCTP (50 p ~ ; 200 cpm/pmol) were substituted for dTTP and [3H]dTTP, respectively. The resulting DNA was purified by Bio-Gel A-5m gel filtration as above.

The 32P-labeled pCM959 and 3H-labeled 6x174 DNAs were pooled and mixed with CsCl to a final density of 1.72 gm/ml. The samples (5 ml) were run in siliconized tubes, pretreated with single-stranded DNA, in a VTi65 rotor a t 55,000 rpm for 24 h at 20°C. Approximately 40 125-p1 fractions were collected from the bottom of the tube, precipitated with trichloroacetic acid onto Whatman 3MM filters, and counted. Recovery was between 70 and 100% of loaded radioactivity (2 X lo4 cpm of 32P; 5 X lo4 cpm of 3H). Parallel gradients for electron microscopy and agarose gel electrophoresis were run without marker DNAs.

RESULTS

Initiation Requires Supercoiled Plasmid DNA-The reconstituted oriC replication system required supercoiled DNA (form I) or the prior action of gyrase on covalently closed relaxed DNA (form IV) (Table I). A more pronounced lag period preceded replication of relaxed DNA (Fig. l ) , during which it became supercoiled. Neither nicked circular (form 11) nor linear (form 111) molecules could serve as replication templates; however, sealing nicked circular DNA with ligase gave it the template properties of relaxed DNA (Table I). As expected, topoisomerase I, known to relax negative supercoils and oppose gyrase action, inhibited replication by extending the lag time (Fig. 1) (5 , 6).

Plasmid replication can be separated into stages of: (i) formation of a prepriming complex, (ii) priming, (iii) elonga-

TABLE I Influence of topology on DNA replication

Reactions (30 pl) were performed with or without dnaA protein, and incubated at 30 "C for 30 min. Preparation of template is described under "Experimental Procedures." "Nicked + ligase" denotes Nicked pCM959 (1 pg) incubated with 0.5 pg of T4 DNA ligase in 66 mM Tris-HC1 (pH 7.6), 5 mM MgC12, 5 mM dithiothreitol, 1 mM ATP (volume = 5 pl) for 4 h at 4 "C and heat-treated (55 "C for 15 min) to inactivate ligase, prior to the addition of replication proteins; agarose gel electrophoresis confirmed that the DNA was converted to the covalentlv closed form.

Template Topology

M13mpRE85, experi- Supercoiled ment 1 Relaxed

M13mpRE85, experi- Supercoiled

pCM959 Supercoiled Relaxed Nicked Nicked + ligase

Nicked

ment 2 Linear

DNA synthesis

+dnaA -dnaA

pmol 366 9 254 6 26 18

245 7 6 7

286 ND" 261 ND 11 ND

291 ND ND, not determined.

= 300

- I

TIME ( m i d FIG. 1. Time course of replication of supercoiled and re-

laxed DNA. SC is supercoiled, or form I M13mpRE85 DNA, and R is covalently closed relaxed, or form IV DNA. Reaction mixtures (210 pl) contained RNA polymerase, and were assembled in the presence or absence of topoisomerase I (Top0 0. DNA synthesis was measured on 30 pl samples taken after incubation at 30 "C.

tion (DNA synthesis), and (iv) termination to produce supercoiled daughter molecules. Three priming systems can oper- ate: a feeble one with RNA polymerase alone, an active one with primase alone, and a somewhat more active one when both are combined ( 5 ) . Initial experiments were performed in the presence of both RNA polymerase and primase (Fig. 1, Table I); subsequently, identical results were obtained with the solo primase system (data not shown). Thus, supercoiling is required in both of these efficient priming systems; the solo RNA polymerase system, which primes very poorly, was not investigated.

Initiation in the solo primase system was separated into stages by manipulating the temperature and protein components. The first stage, formation of the prepriming complex, requires a higher temperature (i.e. greater than 28°C) than subsequent priming and elongation events (6). Typically, the prepriming reaction is carried out at 35-38 "C, followed by priming and elongation at 16 or 24 "C. Before priming by primase can occur, the actions of dnaA, dnal3, dnaC, and HU proteins are required at the elevated temperature; single-

5618 Products of oriC Replication TABLE I1

Influence of topology in the prepriming stage The prepriming reaction on pCM959 DNA contained dnaB, dnaC,

and HU proteins; dnaA protein was present as indicated. DNA synthesis was measured following prepriming, priming, and elongation as described under “Experimental Procedures.” When gyrase was added before the prepriming stage, agarose gels confirmed that the relaxed template was efficiently supercoiled during the incubation.

Topology of Gyrase added before DNA synthesis

pCM959 or after prepriming stage +dnaA -dnaA

pmol Supercoiled Before 94 9

After 89 9 Relaxed Before 95 9

After 9 8

strand DNA binding protein and gyrase can act subsequently at the lower temperature (6).’ Prepriming complex formation with dnaA, dnaB, dnaC, and HU proteins absolutely required supercoiled plasmid (Table 11). Relaxed covalently closed template was not replicated, even though agarose gel electrophoresis confirmed that gyrase efficiently supercoiled the relaxed DNA during the second (low temperature) stage (data not shown). The supercoiling action of gyrase on relaxed DNA was required before the high-temperature, prepriming stage (Table 11). Although dnaA protein can bind nonsupercoiled DNA, albeit with lower affinity (E!), increasing the amount of dnaA protein did not increase the ability of relaxed DNA to replicate (data not shown). It is therefore unlikely that the inability of relaxed DNA to function as a template is due simply to poor dnaA protein binding. Thus, the formation of a prepriming initiation complex requires a negatively supercoiled oriC plasmid.

Initiation Is Rate-limiting; Elongation Is Rapid-Initiation and elongation were usually temporally separated by omitting dNTPs from the first high-temperature incubation so that prepriming and priming were uncoupled from DNA synthesis. Upon addition of dNTPs at a low temperature (16 “C), a burst of DNA synthesis was followed by a slow increase (Fig. 2 4 ) . Nascent, 32P-labeled product strands observed in an alkaline agarose gel (Fig. 2B) were generally of half to full genomic length. Appearance of full-length nascent strands was noted within 1 min; they may be formed as rapidly as 10 s, as is the case for a primed, single-stranded template of comparable length (13). Thus the gradual accumulation of DNA is due to asynchronous initiation, rather than to a slow rate of synthesis. The lag in DNA synthesis in Fig. 1, eliminated by a prior incubation at high temperature, is attributable to a rate- limiting initiation step.

An additional stage of elongation appears at high temper- atures (e.g. ? 30 “C). The initial burst of DNA synthesis is followed by synthesis of products that are greater than unit length (Fig. 2B). This “amplification stage” follows and de- pends on the first elongation stage. For simplicity, elongation was generally carried out under conditions that avoided amplification (e.g. a temperature of 16 “C after initiation at 35 “C in the absence of dNTPs).

Replication with BrdUTP Produces Molecules of Hybrid Density-Buoyant density measurements were made of products formed with BrdUTP in place of dTTP; the rate and extent of DNA synthesis were the same for each nucleoside triphosphate. More than 90% of the newly synthesized DNA banded at the hybrid, or heavy-light, position in CsCl gradients (Fig. 3). Thus, replication is semi-conservative; daugh-

K. Sekimizu, T. A. Baker, and A. Kornberg, unpublished results.

0 5 10 15 TIME (rnin)

(B)

1 6OC 3OoC TIME (rnin) 1 3 5 15 1 3 5 1 5

-WELLS

kb

-6.3

-4.0

.2.9

.2.0

.1.7

.0.3

FIG. 2. Time course of replication in a staged reaction and electrophoretic analysis of the nascent strands. Reaction mixtures (125 pl) were first incubated at 35 “C for 12 min in the presence of all proteins and pCM959 DNA, but in the absence of dNTPs (“Experimental Procedures”). After 2 min at 16 or 30 “C (as indicated), dNTPs were added and 25-p1 samples were analyzed at the indicated times. A, time course of DNA synthesis at 16 and 30 “C. DNA synthesis, measured from 5 pl of each sample, was corrected for the total 25-p1 volume. B, autoradiogram of 32P-labeled strands. Samples (20 pl each) were electrophoresed in an 0.8% alkaline agarose gel (in 30 mM NaOH, 1 mM EDTA), and the gel dried and autoradiographed. Markers were 32P-end-labeled restriction fragments of the indicated sizes (kb = 1000 bp), run on the same gel.


0 10 20 30 40

BOTTOM FRACTION NUMBER TOP

FIG. 3. Buoyant density analysis of DNA replicated with BrdUTP. CsCl gradient profiles of 32P-labeled products of pCM959

covered. 3H-labeled @X174 double-stranded DNA of hybrid (LH) and replication, expressed as the per cent of total labeled product re-

light (LL) density were included in the gradient.

pCM959 M 13oriC5BL2

POLYMERASE - RNA + - +

- 1 -

0.8% AGAROSE 0.6% AGAROSE

FIG. 4. Products of replication of two oriCplasmids. Primase was present in all reactions; 475 ng of RNA polymerase was also included as indicated. Reactions (of 25 pl) were stopped by addition of EDTA (to 10 mM); 5 pl of each sample was acid-precipitated to measure DNA synthesis. The remainder was treated with RNase A (to 10 pg/ml) for 30 min at 30°C followed by the addition of SDS (to 0.25%), and run on neutral agarose gels in 89 mM Tris-borate, 2.5 mM NazEDTA buffer. The SDS treatment removed all protein bound to the DNA because the electrophoretic pattern was identical when deproteinized (by phenol extraction) samples were examined. Left, products of pCM959 run on a 0.8% agarose gel; right, products of M13oriC5BL2 run on a 0.6% agarose gel. The positions of form I (I) and form I1 (ZI) were determined by ethidium bromide staining of unlabeled plasmid markers. The position of 6' form replicative intermediates (see text) is also indicated.

ter molecules contain one new (heavy) strand hydrogen- bonded to one parental (light) strand. Elongation by a rolling circle mechanism, as in phage X late replication (14), or by gap-filling repair synthesis does not significantly contribute to the replication of oriC plasmids. Furthermore, replication was limited to one round/template. In the absence of termination factors needed to produce covalently closed daughter

molecules (see below), the products are not topologically constrained, cannot be supercoiled by gyrase, and are not substrates for initiation.

Products Include 0 Structures and Nicked and Gapped Plas- mids as Monomers and Catenated Pairs-On agarose gels, the 32P-labeled products appeared as multiple bands superim- posed on a smear (Fig. 4). A band migrated as form I1 (nicked and gapped) DNA below a ladder of DNA species, the upper- most of which were the most prominent. Two different oriC plasmids, pCM959 and M13oriC5BL2, showed similar pat- terns except for the shift due to their different sizes. Plasmid pCM959 is 4.0 kb and contains only E. coli sequences neigh- boring oriC; M13oriC5BL2 is 6.7 kb and contains slightly more than the minimal oriC sequence (15) in an M13 vector (see "Experimental Procedures"). The species of products were thus independent of non-oriC E. coli sequences in the template plasmid, and also unaffected by the presence of RNA polymerase (Fig. 4). The pattern of products resembled those observed from SV40 replication in vivo (16-18): (i) individual daughter circles, (ii) multiply intertwined catenated pairs of daughter molecules, and (iii) 8-structure intermediates. The latter were principally "late," or almost complete, and migrated as a dense smear near the top of the DNA ladder (17). In SV40, catenated daughter molecules result from incomplete

(EcoRP) 2nd C- 1 LINEAR D e 1 S t

-e

"D

t - I1

"I

2.9 4.0 6.3 kb

FIG. 5. Two-dimensional gel electrophoresis of replication products. The procedure was modified from Mizuuchi (19). Products of pCM959 replication were run in a neutral 0.8% agarose gel in the first dimension (as in Fig. 4), stained with ethidium bromide, and cut out from the gel. The gel slice was soaked for 2 h in two changes of a buffer containing 150 mM NaCl, 6 mM Tris-HC1 (pH 8), and 0.5 mg of BSA/ml. After warming to 37 "C, the slice was incubated in the above buffer plus 6 mM MgClZ and 350 units of EcoRV for 10 h at 37 "C. After soaking in 89 mM Tris-borate, 2.5 mM NazEDTA, 0.2% SDS for 15 min and the same buffer without SDS for 30 min, the slice was embedded in another 0.8% agarose gel, electrophoresed in this buffer in the second dimension, dried, and autoradiographed. The second dimension was overexposed relative to the first, in order to easily visualize all species. Markers were pCM959 form I and form I1 (unlabeled, visualized after ethidium bromide staining), and 32P- end-labeled restriction fragments of the indicated size (kb = 1000 bp). 6' are 0 structures and D is tentatively identified as displacement loops (see text); in the second dimension 6' and D refer to their positions after restriction digestion.

5620 Products of oriC Replication

FIG. 6. Electron microscopy of replication products. Reaction mixtures containing pCM959 DNA were prepared for electron microscopy as described in the legend to Table 111. A and B are late 0 structures, C is a displacement loop, and D, E, and F are catenated daughter molecules. The bur represents 0.1 pm.

unwinding of helix turns ahead of replication forks prior to termination. These helical turns are transformed to double- stranded intertwines behind the replication forks, so that when DNA synthesis is finished, the daughter molecules remain interwound, or catenated. The number of intertwines between each pair varies depending on the number of Watson- Crick helical turns not removed prior to termination; the resulting molecules have variable topological constraints and migrate as a ladder of DNA bands during agarose gel electrophoresis (17). Two-dimensional gel electrophoresis confirmed a similar interpretation for the products of oriC replication. After digestion with a single-site restriction nuclease, monomer-length circles and molecules that are topologically constrained, such as catenated daughters, should be resolved to unit-length linears, whereas replicative intermediates should migrate more slowly due to greater than unit-length size and to the presence of forked structures. Therefore, a neutral agarose gel of the DNA products (as in Fig. 4) was electrophoresed in a second dimension after linearization with the restriction endonuclease EcoRV in situ (Fig. 5). The nicked and gapped circular DNA (form 11) and the DNA ladder bands were all resolved to unit-length linears in the second dimension. The prominent band or smear (0 on Fig. 5) from the first dimension, however, became a slowly migrating, more diffuse smear after linearization, indicating a structurally complex species, consistent with a heterogeneous population of 0 structures.

Electron Microscopy of the Products-Deproteinized replication products identified by electrophoretic studies were

directly observed by electron microscopy (Fig. 6). Products of a pCM959 replication reaction contained monomer circles (70%), 6' structures (21%), and catenated pairs (9%) (Fig. 6 and Table 111). The number of monomer circles observed included unreacted template molecules because they cannot be distinguished from radiochemically labeled monomeric product molecules by electron microscopy. Product DNA, isolated as the hybrid density fractions from a CsCl gradient of a BrdUTP reaction (Fig. 3; Table 111), contained 0-form intermediates as one-quarter of all structures, which is about one-third of the total mass3; of the remainder, half (as mass) were completed monomers (form 11) and half were catenated pairs.

Most of the replicative intermediates appeared to be near completion with the growing fork (or forks) having traversed most of the plasmid. Even after brief reaction times, early replicative intermediates were relatively scarce. This is consistent with the observation, in Fig. 2, that elongation is rapid; accumulation of intermediates at the late replicative stage implies that termination and segregation steps are slow. Among the 0 structures examined (Fig. 7), only a short length of parental duplex between the growing forks remained unreplicated; 60 of 64 had less than 800 bp (20%) of parental duplex remaining in the intermediate. The average length of unreplicated DNA between the replication forks in these late

The values in Table 111, expressed as per cent of structures, were converted to per cent of mass by assuming that late 0 structures and catenated pairs had twice the mass of monomer structures.


TABLE I11 Electron microscopy of replication products

SDS-treated pCM959 reaction mixtures (“Total reaction”) were deproteinized by Bio-Gel A-5m filtration. Void-volume fractions were pooled, dialyzed into 20 mM NaCl, 5 mM Na2EDTA, and spread for electron microscopy essentially as described by Schnos and Inman (20). Product DNA was identified as the pooled heavy-light, or “hybrid-density” fractions from CsCl gradient analysis of BrdUTP- labeled pCM959 (as in Fig. 3). Samples were analyzed in a Philips EM300 at 40 kV. Only circular structures were scored; catenated pairs were counted as one structure and included all circles that overlapped, even if significant intertwining was not evident. (Spread- ings of monomer plasmid preparations at least 10-fold higher in concentration showed less than 3% overlapping circles.) 0 structures (“Total”) included those with two double-stranded daughter arms (“Double-stranded”), and with one single-stranded and one double- stranded arm (“Disdacement loom”).

Structure Total Hybrid-density reaction fractions

% of total Monomer 70 47 0 structures

Total 21 27 Double-stranded 15 ND” Displacement loops 6 ND

Catenated pairs 9 26

Number counted 212 130 ND. not determined.

0 1000 2000 3000 4000 LENGTHOFPARENTALDUPLEX

IN 8 INTERMEDIATES (bp)

FIG. 7. Extent of replication of 0 structures. Micrographs of 64 pCM959 0 structures were measured on a Summagraphics ID 2000 series Digitizer interfaced to an IBM-XT computer. The length of unreplicated duplex parental DNA between replication forks was determined as a fraction of total molecular length (4012 bp) and converted to base pairs.

intermediates was 220 f 160 bp, or about 5% of the molecular length.

More than two-thirds of replicative intermediates were double-stranded on both arms of the replication bubble. The remainder were displacement loops (Fig. 6C) in which synthesis of only the leading strand had occurred; in most of these the growing fork had moved almost completely around the circle. Thus, DNA synthesis can occur on both parental DNA strands, but does not always. The existence of displacement loops, especially when extensive, indicates that leading strand replication can occur without concomitant lagging strand synthesis in these reactions. The products of M13oriC5BL2 replication were similar, indicating these structures are not particular to the oriC plasmid construction. This reconstituted reaction may be deficient in factors necessary for efficient initiation of both strands, inasmuch as displacement loops among replicative intermediates in crude enzyme systems are rare (less than 2%).

Late displacement loops, an unusual intermediate, cannot be unambiguously identified in agarose gels; their single- strandedness probably results in a faster migration than dou-

ble-stranded 0 structures. Band D (Fig. 5) , one of the “rungs” of the DNA ladder, is a possibility. EcoRV digestion failed to resolve this species into unit-length linear molecules, but instead created a smear similar to that of late 0 forms in the second gel dimension (Fig. 5). As the only other major complex species seen by electron microscopy, this band likely corre- sponds to the late displacement loop molecules.

Electron microscopy of reaction products of amplified synthesis contained no novel forms of unit-length molecules (data not shown), but rather very long and complex branched DNA molecules which were not examined in detail.

Termination Requires pol I and Ligase, and Is Stimulated by RNase H-Due to a lack of enzymes needed to excise primers or ligate DNA strands, covalently closed products were not observed in the replication reactions described above (Figs. 4 and 5). DNA polymerase I (pol I), RNase H, and E. coli ligase were therefore added to the reaction as termination enzymes. Termination, defined as the final processing events required to make supercoiled daughter molecules, was separated temporally from elongation by labeling products with [32P]dTTP during elongation and adding excess unlabeled dTTP along with the termination enzymes. The assay thus measured termination of products made during the pulse.

Two supercoiled species appeared when pol I, ligase, and RNase H were present (Fig. 8, right lane). One was clearly

Pol I - - + - + - + LIGASE - - - + + + + RNase H - + - - - + +

“I

FIG. 8. Enzyme requirements for termination. Termination was separated temporally from elongation using pulse-chase experiments. Replication reactions were performed using pCM959 DNA as described under “Experimental Procedures,” except the concentrations of dNTPs during elongation (the pulse) were: [32P]dTTP, 20 PM; dATP, dCTP and dATP, 100 p~ each. After 10 min at 16 “C, unlabeled dTTP was added to 0.5 mM, and the indicated enzymes added in the following amounts: DNA polymerase I, 25 ng; RNase H, 0.14 ng; and E. coli ligase, 3 ng. The assay included 80 p~ NAD and 5 mM NH&l for ligase activity. After another 20 min at 16 “C, the reactions were stopped, DNA synthesis was measured, and samples were analyzed by agarose gel electrophoresis and autoradiography. DNA synthesis after the elongation stage was 107 +. 3 pmol in all samples. 32P-labeled DNA species are labeled as previously identified (Figs. 4 and 5). A new band or smear appeared in the presence of pol I + ligase f RNase H, and was identified as supercoiled catenated pairs (SC catenanes), see text.


40

30

9 20 n

Y 10 n

z a u

U 0 I- 2

K e u1

s 20

10

0 0 5 10 15 20 BOTTOM FRACTION NUMBER TOP

FIG. 9. Alkaline sucrose gradients of pulse-labeled replication products. Pulse-chase reactions with pCM959 DNA (%fold, 50 pl) were performed in the absence (A) and presence (E) of pol I, RNase H, and ligase (as in Fig. 8), stopped with NazEDTA (to 20 mM) and SDS (to 0.5%), mixed with an equal volume of 1 M NaOH for 10 min at 25 "C, and loaded onto a 1.95-ml5-20% alkaline sucrose gradient over a 60% cushion (in 0.1 M NaOH, 0.9 M NaC1). The gradient was centrifuged in a TLS55 rotor in a Beckman TLlOO ultracentrifuge for 75 min at 55,000 rpm and 12 "C. Approximately 19 2-drop (-100 pl) fractions were collected from the bottom of the tube, acid-precipitated as for DNA synthesis aliquots, and counted. The ordinate is the per cent of total radioactivity recovered from the gradient (90-100% of the lo5 cpm loaded). The position of nascent single-stranded linears and circles (SS) was determined from both agarose gels and parallel gradients of form I1 3H-labeled M13oriC5BL2 DNA. The position of form I pCM959 was identified by agarose gel electrophoresis of the gradient fractions. The fast- migrating fractions consisted of heterogeneous supercoiled species, monomers, and multiply intertwined catenated daughters, producing a broad peak.

TABLE IV Enzyme requirements for termination

Pulse-chase experiments were performed as described in the legend to Fig. 8. Supercoiled product was measured as the per cent of fast- sedimenting material in alkaline sucrose gradients (Fig. 9). Approxi- mately IO5 cpm of 32P were loaded onto each gradient; recovery was always greater than 90%. The values for ligase only and RNase H plus ligase, although low, were statistically above background.

Enzymes added during the Supercoiled chase product

% None <0.2 Ligase 0.8 RNase H + ligase 1.8 Pol I + ligase 14 Pol I + RNase H + ligase 24

identified as supercoiled monomer plasmid (form I). The other was a smear near the bottom of the ladder, and is probably made up of supercoiled catenated dimers, inasmuch as such structures occupy the same position as supercoiled SV40 catenated dimers relative to supercoiled and nicked monomers (17), and the DNA ladder (the nicked catenated daughters found in the absence of termination enzymes) was less prev- alent. The identification was confirmed by two-dimensional

gel electrophoresis: after EcoRV digestion, this smear migrated as unit-length linears in the second dimension. When the first dimension gel slice was denatured in situ (without restriction digestion) and run in alkaline agarose in the second dimension, the smear migrated more slowly than unit-length linears, consistent with a topologically constrained, but larger than unit-length, species (data not shown).

None of the three termination enzymes can alone produce topologically constrained species. Termination required both DNA polymerase I and ligase and was stimulated by RNase H. Gyrase, present throughout the reaction, very efficiently supercoils daughter molecules once they are ligated (topologically constrained). Quantitative measurement of the supercoiled species from the autoradiograms was difficult because supercoiled catenated molecules migrated with unfinished products. Alkaline sucrose gradients were used to separate nascent products from topologically constrained molecules which sediment more rapidly (Fig. 9). After a 20-min chase, about 25% of the products were supercoiled when pol I, ligase, and RNase H were present (Table IV). No detectable supercoiled species were measured in the absence of all three enzymes; however, low, but measurable levels were seen with ligase only or RNase H plus ligase (Table IV). This may be due to pol I contaminating some of the replication enzyme preparations, or to DNA polymerase I11 repair synthesis of gapped form I1 molecules during the pulse. pol I and ligase were required for significant levels of termination, and were stimulated by RNase H. Very small amounts of these enzymes were required for maximal termination activity. The experimental molar ratios of enzyme:input template were 6:l for pol I, 1:lO for RNase H, and 1:2 for ligase. The stimulation by RNase H was not replaced by additional amounts of pol I.

During the chase period, the products made during the pulse were converted to supercoiled forms (Fig. lo), but termination was slow relative to elongation. About 30% of the products were supercoiled after 30 min at 16 "C. The catenated pairs were eventually decatenated, presumably by gyrase, into supercoiled monomers (form I).

The delay in termination is probably caused by slow replication of the last 200 or so base pairs. Buildup of late 6' structures (Fig. 7) implies that the growing forks move very slowly through this region. This may reflect steric hindrance by proteins bound to the DNA or by the slow unwinding of the last few helical turns between the growing forks. Chase of late 6' structures into the other products identifies them as true intermediates rather than dead-end products. The slow decatenation (Fig. 10) may reflect suboptimal conditions for gyrase, such as salt concentrations that exceeded the optimum for its decatenation activity (21).

DISCUSSION

Template Topology Is Important during Initiation-For- mation of a prepriming complex with dnaA, dnaB, dnaC, and HU proteins on oriC DNA absolutely requires a supercoiled template. The additional requirement for high temperature during this stage (6) suggests that localized helix melting and changes in DNA conformation may be involved. The com- plexity of the origin sequence has led to proposals that sec- ondary structure is important for initiation (22-24). The binding of dnaA protein alone is enhanced by, but not de- pendent on supercoiling (12). The dnaA protein-oriC complex contains many molecules of dnaA protein around which the DNA is wrapped in some specific way (25). One of the func- tions of dnaA protein, besides oriC recognition, may be to facilitate or stabilize a specific DNA conformation required for assembly of an active prepriming complex. The require-


TIME (min) 0 10 20 30 60

”I

0 20 40 60 TIME (mid

FIG. 10. Time course of termination. A, autoradiogram of a pulse-chase experiment using pCM959 DNA. Elongation with [“PI dTTP was for 6 min at 16 “C, followed by a chase for the indicated times with a 25-fold excess of unlabeled dTTP, pol I, RNase H, and ligase as described in the legend to Fig. 8. B, appearance of supercoiled product (SC). The amount is expressed as the per cent of [3zPpl label in the fast-sedimenting material of alkaline sucrose gradients (run as in Fig. 9) of each sample. Monomer (form I) DNA was quantitated, relative to radioactivity, by densitometry of the autoradiogram in A.

ment for supercoiling explains why topoisomerase I, via its relaxing activity, is so inhibitory (Fig. 1) (5,6). An activating role of RNA polymerase is inferred from the lowering of the temperature required for prepriming (6); presumably transcription helps open the helix.

Supercoiled Daughter DNA Molecules Are Identical in Se- quence and Topology to Parental Molecules-At least a dozen purified proteins are required for the several stages of DNA synthesis: (i) prepriming complex formation, (ii) priming, (iii) elongation, and (iv) termination of the newly synthesized strands.

The replication reaction reconstituted in vitro is authentic by several criteria: dependence on the oriC sequence, dnaA and other replication proteins (4-6), and the synthesis of authentic products. Replicative intermediates are 0 structures, miniature versions of the replicating E. coli chromosome. The daughter molecules are circles with one newly synthesized

strand hydrogen-bonded to one parental strand. Based on the microscopic and electrophoretic studies, in-

dividual and catenated daughter molecules are the predomi- nant products. Late 0 structures accumulate because the termination and segregation steps proceed much more slowly than elongation. Some of the 0 structures are actually displacement loops, replication having copied only one of the parental template strands. It is uncertain whether these structures are eventually converted to two double-stranded daughter molecules, to one double- and one single-stranded circle, or remain as dead-end intermediates. Single-stranded circles, either individual or half of a catenated pair, were not seen by electron microscopy ( ~ 0 . 2 % of circular structures). If nicked or degraded, they could not be distinguished from heterogeneous residual single-stranded material persisting in the plasmid preparations. This seems unlikely, however, because displacement loop single-stranded arms were intact. The band tentatively identified as displacement loops (Fig. 5) decreases with time (during the pulse-chase experiment in Fig. lo), implying that they (as well as 0 structures) are intermediates rather than dead-end forms.

Production of completed covalently closed daughter molecules requires further processing: excision of RNA primers, filling the resulting gaps and sealing nicks. Genetic and en- zymological studies have implicated DNA polymerase I (pol I), RNase H, and DNA ligase in the termination process. Mutants in pol I (polA) and in ligase (lig) accumulate short (<lo S) nascent replicative fragments (26-29); RNase H (rnh) polA double mutants accumulate more short fragments than polA alone (30). Because E. coli ligase cannot ligate DNA to RNA (31), all RNA residues must be removed and replaced with DNA. DNA polymerase I11 holoenzyme has no 5‘+3‘ exonuclease activity to excise RNA (32, 33). Primers can be removed by both RNase H and pol I, but only pol I can efficiently remove the last RNA residue (34, 35). In our studies, pol I was required to produce daughter molecules that were suitable substrates for ligase; RNase H stimulated the process. RNase H involvement in removal of long RNA primers in vivo (30, 36) may also extend to the short primers presumed to be present in this in vitro system. The essential role of pol I seen here is consistent with the phenotype of polA mutants and the absence of null polA mutants, specifi- cally those completely deficient in 5‘+3‘ exonuclease activity.

Catenated daughter molecules predominate among the early products, and are slowly decatenated to monomer plasmids. Yet it seems unlikely that plasmids, or the E. coli chromosome, are catenated to a significant extent. The inefficiency of completion and of “segregation” of late 0 structures may be due to the lack of another protein (or proteins), absent DNA sequences, or to unfavorable incubation conditions. Although proper terminations are observed in our system, the events surrounding the terC termination region of the E. coli chromosome are probably more complex (37).

In our reconstitution of the complete cycle of oriC plasmid replication, beginning and ending with supercoiled molecules, only about one-fifth of the available template molecules are used for replication, and of those, only one-third are completed. It is not known whether one or two replication forks are active on each template. The presence of unit-length nascent strands (Fig. 2) indicates that in some cases only one fork has traversed the entire circle. Other replication proteins (e.g., n, n’, nn, and i) may be required for efficient coupling of leading and lagging strand synthesis. In addition, several partially purified proteins are known to stimulate replication (4) and may contribute to an optimal system of oriC replication.


Acknowledgments-We thank J. D. Griffith for kindly providing 17. Sundin, O., and Varshavsky, A. (1981) Cell 25,659-669 computer programs to measure molecular length, and D. L. Brutlag 18. Weaver, D. T., Fields-Berry, S. C., and DePamphilis, M. L. (1985) for assistance with program modification. We are grateful t o LeRoy Cell 41, 565-575 L. Bertsch for assistance in preparing this manuscript. 19. Mizuuchi, K. (1984) Cell 39, 395-404

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