INTRODUCTION

Investigations of the biochemical mechanisms involved in the initiation of bacteriophage  DNA replication have been aided by the reconstitution of the initiation reaction with a defined set of purified  and Escherichia coli proteins (1, 2). These studies have demonstrated that an ordered series of nucleoprotein structures are assembled at ori, the viral replication origin, and subsequently partially disassembled during the establishment of the apparatus responsible for replication fork propagation (2-5).

The first step in the initiation pathway consists of the binding of multiple copies of the  O protein to the viral origin and the subsequent self-assembly of this replication initiator into a nucleoprotein structure called the O-some (6, 7). The O-some both localizes the origin and serves as the foundation for the assembly of more complex nucleoprotein structures. Simultaneously, in solution, several molecules of the  P protein bind to the hexameric E. coli DnaB helicase, to form a P·DnaB complex (8-10). One or more molecules of this latter complex binds to the O-some, as a consequence of specific protein-protein interactions between O and P, to form a second stage nucleoprotein structure containing O, P, and DnaB. Protein-protein interactions within this ori·O·P·DnaB structure prevent release and activation of the helicase activity of the DnaB molecule or molecules present in the complex (7). Binding of the E. coli DnaJ protein to the ori·O·P·DnaB preinitiation structure (3) sets the stage for the ATP-dependent partial disassembly of the complex by the host DnaK (Hsp70) molecular chaperone (4, 5, 11), a reaction that is aided by the GrpE protein (2, 4). This protein turnover reaction results in the release of a portion of the bound P and DnaJ proteins, as well as the liberation of DnaB and activation of its intrinsic helicase activity (4, 5, 11, 12). If the ori template DNA is sufficiently negatively supercoiled, the released DnaB is inserted between the two DNA strands, presumably at the A + T-rich region of ori (7, 12, 13), and becomes active as a replicative helicase.

The transfer of DnaB helicase onto the DNA template completes the initiation phase of  DNA replication and triggers a second series of protein-protein and protein-DNA interactions that culminate in assembly of the replication fork apparatus. The precise assembly pathway remains to be defined, but it is believed to include the following course of events. Single-stranded DNA (ssDNA)¹ created by DnaB helicase action is stabilized by the binding of stoichiometric quantities of the E. coli single-stranded-DNA binding protein (SSB) (3, 12, 14). The E. coli primase (DnaG protein) binds transiently to the DnaB helicase (15, 16) and subsequently synthesizes short RNA chains that serve as primers for synthesis of the leading and lagging DNA strands by the DNA polymerase III holoenzyme (DNA pol IIIh) (3, 15).

Biochemical studies of  DNA replication in vitro have demonstrated that detectable levels of O, P, and DnaK remain associated with the ori template DNA following the chaperone-mediated partial disassembly reactions that bring about initiation of  DNA replication (4, 11, 17). It is still uncertain if one or more of these proteins is associated with the  replication fork apparatus. Although none of the evidence available to date suggests a direct role for the  O and P replication proteins or the DnaK/DnaJ/GrpE chaperone system in the propagation of replication forks during  DNA replication (4), there remains a possibility that one or more of these proteins that act during the initiation phase of the reaction have an auxiliary role during the fork movement phase.

We wished, therefore, to characterize the properties of replication forks established by the  O and P proteins for comparison to the behavior of replication forks established with E. coli replication proteins. To study replication fork assembly and movement, we adapted the rolling circle replication assay pioneered by Lechner and Richardson (18), in which a tailed, nicked circular (TNC) DNA molecule serves as the replication template. With this assay, we can take advantage of the capacity of the  O and P proteins to mediate the transfer of DnaB helicase onto any ssDNA, even if the ssDNA is stoichiometrically coated with SSB (19, 20). A primary benefit of the rolling circle replication system for studies of the propagation of  replication forks is that there are no topological constraints to encumber fork movement. This is in sharp contrast to the situation encountered with the standard ori plasmid replication assay, in which the rate of fork movement is probably limited by the rate at which topoisomerases relax the positive supercoils generated during replication of a covalently closed, circular template (21, 22).

Our studies demonstrate that the  O and P replication proteins efficiently mediate the formation of robust replication fork assemblies. Such " replication forks" synthesize greater than 100 kb of DNA at rates approaching 750 nucleotides/s at 30 °C. This rate is indistinguishable from the rate of movement of replication forks established by a minimal two-protein system composed of DnaB helicase and DNA pol IIIh.

EXPERIMENTAL PROCEDURES

The sources of materials were as follows: AMP-PNP, Sigma; Hepes, Research Organics; bovine serum albumin (BSA; fraction V) Miles Laboratory Inc.; unlabeled ribonucleoside triphosphates (rNTPs), unlabeled deoxyribonucleoside triphosphates (dNTPs), dT₂₀₀ oligonucleotide and DEAE-Sephacel, Pharmacia Biotech Inc.; [methyl-³H]dTTP (40-70 Ci/mmol), ICN; [-³²P]dCTP (~800 Ci/mmol) and [-³²P]ATP (>5000 Ci/mmol), Amersham Corp.

The buffers used were as follows: TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA); core buffer (10 mM Tris-HCl, pH 7.5, 10 mM MgCl₂, 100 µg/ml bovine serum albumin); high salt gradient buffer (50 mM Tris-HCl, pH 8.0, 0.7 M NaCl, 1 mM EDTA); neutral agarose-gel sample buffer (0.5% bromphenol blue, 60% (w/v) sucrose, 0.5% (SDS)); alkaline gel sample buffer (0.5% bromcresol green, 0.25 M NaOH, 10 mM EDTA, 0.5% SDS, 60% sucrose, and ³²P-labeled, linear pEMBL130(+) plasmid DNA (750 cpm/gel sample)); X EDB (25 mM Tris-HCl, pH 7.5, 10 mM dithiothreitol, 100 µg/ml BSA, 5% sucrose); buffer A (50 mM Tris-HCl, pH 7.5, 20% (v/v) glycerol, 1 mM EDTA, 1 mM dithiothreitol, 5 mM MgCl₂, 0.1 M NaCl); TGE buffer (25 mM Tris, 0.19 M glycine, 1 mM EDTA); and S1 nuclease buffer (50 mM sodium acetate, pH 4.8, 0.5 M NaCl, 1 mM ZnCl₂, 30 µg/ml denatured calf thymus DNA).

The E. coli K-12 strains used and their relevant genetic characteristics were as follows. SK6776 (uvrD288) is a DNA helicase II mutant (provided by Dr. C. McHenry, University of Colorado School of Medicine); C600dnaK103/pJK23 (23) is a GrpE protein overproducer (provided by Dr. C. Georgopoulos, University of Geneva); 71-18 (F) and 71-18/pEMBL130(+) (24) were used for propagation of M13 phage derivatives (provided by Dr. G. Cesareni, University of Tor Vergata (Rome)); RLM727 (HfrH/pRLM55) is a thermoinducible strain for amplification of E. coli SSB that was constructed in this laboratory (25); RLM861 (N100/pRLM31) is a recA, thermoinducible, DnaB-overproducing strain that was constructed in this laboratory.² Strain RLM973, which is 71-18/pRLM85, is described in this paper.

DNA and /HindIII DNA size standards were purchased from New England Biolabs, Inc.; T7 DNA was purchased from the U.S. Biochemical Corp. Phage M13mp19 was a gift of Drs. T. Seeley and L. Grossman of this department. /KpnI and linear pEMBL130(+) DNA size standards were prepared by digesting these DNAs with KpnI and BamHI, respectively, followed by treatment with calf intestine phosphatase. Protein was removed by phenol extraction, and the DNAs were concentrated by ethanol precipitation. The plasmid pRLM85 was constructed by E. Hwang in this laboratory by cloning a Sau3AI fragment of DNA (positions 38815-39577; Ref. 26), containing the  origin of DNA replication, into the BamHI site of pEMBL130(+) such that the R (lower) strand of ori is inserted into the (+)-strand of the plasmid. M13mp19oriL was constructed by cloning from pRLM85 a 0.8-kb PstI to SacI fragment, containing ori and the majority of the pEMBL130(+) polylinker sequence, into M13mp19 DNA that had been digested with both PstI and SacI, which removed most of the mp19 polylinker sequence. Phage M13K07 (27) was a gift of Drs. E. Hildebrand and L. Grossman of this department. Oligodeoxyribonucleotides were constructed by automated solid phase synthesis by S. Morrow of this department and were used without further purification. Oligonucleotide RM34 is 5-CGGACCTGCAGGCAT-3; it is complementary to (+)-strand DNA at the position of the unique PstI site of M13mp19oriL. Oligonucleotide RM35 is 5-AATTCGAGCTCGATAT-3; it is complementary to (+)-strand DNA at the position of the unique SacI site of M13mp19oriL.

PstI, SacI, KpnI, and BamHI restriction endonucleases and T4 polynucleotide kinase were from New England Biolabs. Calf intestine phosphatase, proteinase K, and DNase I were from Boehringer Mannheim. S1 nuclease was from Pharmacia. Homogeneous E. coli UvrD protein (15,000 units/mol) (28) was a generous gift of Drs. Jaya Yodh and Randy Bryant of this department. Antibody directed against E. coli UvrD protein was graciously donated by Drs. Robert Lahue and Paul Modrich (Duke University). The -subunit of the E. coli DNA pol IIIh was kindly provided by Dr. Mike O'Donnell (Rockefeller University).

All  and E. coli replication proteins used were estimated to be greater than 95% pure. Purification and specific activities of the  O and P replication proteins and the E. coli DnaJ, DnaK, DnaG primase, and DNA polymerase III holoenzyme proteins have been described previously (1). Some preparations of DNA pol IIIh (3 × 10⁵ units/mg) were purified from SK6776 (uvrD) essentially as described (29). DNA polymerase III* was purified by a modification of the published protocol (30), using a procedure developed by Dr. M. O'Donnell (Rockefeller University). E. coli SSB (7 × 10⁴ units/mg) was purified from strain RLM727 (HfrH/pRLM55) by a modification of the protocol of LeBowitz (25). Briefly, the blue dextran-Sepharose column was replaced by a ssDNA cellulose column prepared as described by Alberts (31). Approximately 57.5 mg of fraction II protein (>95% SSB) was diluted with buffer A (minus NaCl) to a conductivity equivalent to buffer A and applied at 3 column volumes/h to an ssDNA cellulose column (150 ml, 5.75 mg ssDNA/ml) that had been equilibrated with buffer A. The column was washed with buffer A containing 0.25 M NaCl until the absorbance at 280 nm approached zero. Bound SSB was eluted with buffer A containing 2 M NaCl and purified further by chromatography on hydroxyapatite as described (25). E. coli GrpE protein (6.4 × 10⁵ units/mg) was purified essentially as described by Zylicz et al. (32), except that the strain used was C600dnaK103/pJK23 (23).

E. coli DnaB protein was purified from 300 g of thermally induced RLM861 by a modification² of the protocol of Ueda et al. (33). Following the 0.24 ammonium sulfate and two 0.20 ammonium sulfate backwashes of the 0-40% ammonium sulfate precipitate, the residual protein precipitate was resuspended in 18 ml of buffer A (fraction II, 27 ml, 1070 mg, 4.7 × 10⁷ units). A 4-ml portion of fraction II (160 mg of protein) was diluted 3-fold with buffer A and applied to a 75-ml ATP-Sepharose column that had been prepared by the technique of Lamed (34), except that the amount of adipic dihydrazide used as a linker ligand was reduced 10-fold to 0.9 g/100 ml. This modification resulted in lowered amounts of ATP covalently linked to the Sepharose matrix (typically 1 µmol of ATP/ml of gel matrix), a reduction that correlated with improved stability of DnaB protein eluted from the ATP affinity column.³ The ATP-Sepharose column was washed with 5 column volumes of buffer A, followed by 10 column volumes of buffer A containing 10 mM MgCl₂ and 10 mM AMP. DnaB protein was subsequently eluted with buffer A with 20 mM sodium pyrophosphate (fraction III, 11 mg, 2.3 × 10⁶ units). Fraction III was applied to a 7-ml DEAE-Sephacel column that had been equilibrated with buffer A. The column was washed with 5 volumes of buffer A containing 0.15 M NaCl, followed by 2 column volumes of buffer A containing 0.2 M NaCl. DnaB was eluted with 5 column volumes of buffer A containing 0.4 M NaCl (fraction IV, 6 mg, 3.4 × 10⁶ units).

The plasmid pEMBL130(+) was propagated in the ssDNA form by superinfection of 71-18/pEMBL130(+) cells with the helper phage M13K07 by the method of Dente et al. (27). M13mp19oriL was propagated in the E. coli strain 71-18; phage particles were isolated from the cell-free supernatant as described (35). Phage DNA was isolated free of contaminating protein and RNA by using a modification of the plasmid isolation protocol of Birnboim and Doly (36). Phage prepared from a 1-liter culture of infected E. coli, as described above, were resuspended in 5 ml of TE buffer and denatured by the addition of 10 ml of a solution containing 0.2 M NaOH and 1% SDS. Contaminating protein and RNA was precipitated by the addition of 7.5 ml 3 M sodium acetate, pH 4.8. Following centrifugation, the DNA remaining in the supernatant was precipitated by an equal volume of ethanol. The pEMBL130(+) ssDNA prepared by this technique is suitable for digestion with restriction enzymes.

For some preparations of the tailed rolling circle DNA template, the circular single-stranded pEMBL130(+) plasmid DNA was isolated free of helper phage DNA by sucrose gradient centrifugation. The DNA mixture was diluted with high salt gradient buffer and layered onto an 11-ml 5-30% sucrose gradient prepared in high salt gradient buffer (each tube contained a 1-ml cushion of 50% sucrose). The DNA samples were centrifuged in a Beckman SW41 rotor at 25,000 rpm for 16 h at 20 °C. Fractions (0.5 ml) were collected from the bottom, and portions of each fraction were analyzed for the presence of DNA by neutral agarose gel electrophoresis. The fractions free of helper phage DNA were pooled, and the pEMBL130(+) DNA was concentrated by ethanol precipitation. The DNA sample (350 µg) was resuspended in 0.5 ml of TE buffer, adjusted to 0.1 M NaOH and applied to a 12-ml Bio-Gel A-15 M column that had been equilibrated with 0.1 mM Tris base. Fractions (0.5 ml) were collected and subsequently analyzed by neutral agarose gel electrophoresis. The fractions containing the peak of pEMBL130(+) ssDNA were pooled, neutralized by the addition of volume of 1 M Tris-HCl, pH 8, and precipitated with ethanol. The precipitate was collected by centrifugation and resuspended in TE buffer, and the purified circular ssDNA was used for the preparation of tailed DNA circles.

The ssDNA 800-nucleotide primer (800-mer) used to form the 5-tail of the TNC DNA template and helicase substrate was prepared by digesting ssDNA at restriction sites made duplex by the presence of hybridized oligonucleotides. 500 µg of mp19oriL ssDNA was suspended in 3.2 ml of core buffer and mixed with 42.3 µg of oligonucleotide RM34, 17.3 µg of oligonucleotide RM35, PstI (600 units), and SacI (800 units); the mixture was incubated at 37 °C for 60 min. The 800-mer was isolated free of linear M13mp19 DNA by neutral agarose gel electrophoresis and was recovered from the gel by electroelution using a Schleicher and Schuell Elutrap device. Some preparations of the 800-mer were treated with calf intestine phosphatase before purification by gel electrophoresis. This permits efficient end labeling of the linear strand of the helicase substrate/rolling circle DNA template by T4 polynucleotide kinase for preparation of a labeled size marker.

Hybridization of the 800-mer to the pEMBL130(+) ssDNA (the 800-mer contains at its 3 terminus a 42-nucleotide-long sequence that is complementary to the pEMBL130(+) polylinker) was accomplished by mixing 250 µg of circular ssDNA with 25 µg of the 800-mer in TE buffer containing 0.5 M NaCl in a final volume of 1.25 ml. The mixture was incubated at 65 °C for 2 min and then immediately supplemented with 1.37 mg of E. coli SSB and incubated for an additional 60 min at 37 °C. The final DNA concentration was 0.14 mg/ml and the molar ratio of pEMBL130(+) ssDNA to 800-mer was 2:1. The hybrid was diluted 7.67-fold into X EDB containing 2.5 mM ATP, 13 mM MgCl₂, 2.5 mM spermidine chloride, 100 µM each dATP, dCTP, dGTP, and dTTP, 16 µg of DNA pol IIIh -subunit, 81 µg of SSB, and 67,500 units of DNA polymerase III* (X EDB replication mix). Some template batches were prepared using partially purified DNA pol IIIh ~(2 × 10⁵ units/mg; Ref. 29) instead of DNA polymerase III* and -subunit. DNA synthesis was carried out during a 30-min incubation at 30 °C and was terminated by chilling the mixture to 0 °C. In some instances, radiolabeled TNC DNA template was prepared. On these occasions, the replication mixture contained [³H]dNTPs or [-³²P]dNTPs at approximately 100-200 cpm/pmol of dNTP. The TNC DNA product was concentrated 10-fold using an Amicon Centricon 30 device. Replication proteins and unreplicated pEMBL130(+) ssDNA were removed from the TNC DNA template by sucrose gradient centrifugation. The DNA sample was supplemented with SDS to 1% (w/v) and diluted with 0.25 volume of 5 × high salt gradient buffer. Portions were layered onto neutral 5-30% sucrose gradients and centrifuged and analyzed as described above. The fractions containing double-stranded DNA were pooled, and the TNC DNA product was concentrated by ethanol precipitation.

For those instances in which helper phage DNA was first removed from pEMBL130(+) ssDNA by sucrose gradient centrifugation before synthesis of the TNC DNA template, a simplified protocol was used for synthesis and purification of the template. The 800-mer was mixed with pEMBL130(+) ssDNA at a molar ratio of 1.5 (800-mer:circle) and hybridized as described above. Once formed, the DNA hybrid was diluted into X EDB replication mix as described above, except that the mix contained 100,000 units of DNA pol IIIh instead of DNA polymerase III* and -subunit. The TNC DNA replication product prepared in this manner had negligible amounts of contaminating single-stranded DNA. The TNC DNA sample was incubated for 4 h at 55 °C with proteinase K (1 mg/ml in 0.5% N-lauroylsarcosine) and extracted three times in succession with neutralized phenol/chloroform/isoamyl alcohol (24:24:1). DNA product in the aqueous phase was precipitated with ethanol, resuspended in TE buffer, and used directly as a substrate/template in rolling circle DNA replication assays.

DNA Replication Fork Movement Assays (Rolling Circle Replication (RCR) Assay and  RCR Assay)

The reaction mixture (25 µl) for the standard RCR assay (also denoted the minimal RCR system) contained 50 mM Hepes-KOH, pH 7.6, 10 mM magnesium acetate, BSA (200 µg/ml), 10 mM dithiothreitol, 2.5 mM ATP, 42 fmol TNC DNA, 100 µM concentration each of dATP, dCTP, dGTP, and TTP with [-³²P]dCTP at a specific activity of 100-2500 cpm/pmol total deoxynucleotide or 172 µM concentration each of dATP, dGTP, and dCTP and 72 µM dTTP with [methyl-³H]dTTP at a specific activity of 70-100 cpm/pmol total deoxynucleotide, 200 units DNA pol IIIh, 0.043-1.9 µg (0.14-6 pmol, as hexamer) DnaB protein, and, where indicated, 2.2 µg of SSB. Typically, the reaction mixtures for rolling circle DNA replication were assembled at 0 °C; following the addition of dNTPs and DNA pol IIIh, DNA synthesis was initiated by a 30-min incubation at 30 °C. In some experiments, however, DnaB was mixed with the TNC DNA template and 200 µM AMP-PNP at 0 °C, and the mixture was subsequently incubated at 30 °C for 5 min to stimulate binding of DnaB to the ssDNA tail of the TNC template. The mixture was then returned to 0 °C, supplemented with the other reaction components, including ATP, and incubated at 30 °C for 30 min.

In certain other experiments, a third approach (denoted the  RCR assay or system) was used to load DnaB onto the TNC DNA template. DnaB was assembled on the ssDNA tail of the TNC template using the phage and host proteins required for initiation of bacteriophage  DNA replication (1, 20). In these instances, the standard reaction mixture (25 µl) contained: 53 ng of  O, 80 ng of  P, 86 ng of DnaB, 144 ng of DnaJ, 6.9 µg of DnaK, 80 ng of GrpE, 200 units of DNA pol IIIh, and, where indicated, 2.2 µg of SSB. To repress the intrinsic functional activities of DnaB (10),  P and DnaB were mixed together (at a 2:1 molar ratio of P to DnaB) before the addition of the DNA template and other proteins. Reaction mixtures were assembled at 0 °C, and rolling circle DNA synthesis was initiated by warming the reaction mixture to 30 °C. For all types of rolling circle replication reactions, DNA synthesis was terminated by chilling the reaction mixture to 0 °C. However, if the DNA replication reaction mixtures were to be analyzed for the sizes of newly synthesized DNA chains by electrophoresis in an alkaline agarose gel, DNA synthesis was terminated by the addition of EDTA to a final concentration of 50 mM. To conserve TNC DNA template, the reaction volumes and components were reduced by half in certain experiments. This had no detectable effect on the properties of the system. In these cases, the DNA synthesis values obtained were normalized (multiplied by 2) to permit direct comparison with results obtained with standard reactions.

The amount of DNA synthesis was usually determined by measuring the level of labeled deoxynucleotide incorporated into trichloroacetic acid-precipitable material, which was collected by filtration through glass fiber filters (Whatman, 934-AH) and then washed, dried, and counted in a liquid scintillation spectrometer as described previously (1). BSA (200 µg) was added as a carrier to all reaction mixtures, before the addition of trichloroacetic acid, to aid precipitation of DNA. If the reaction volume was limiting, however, the amount of DNA synthesis was determined by spotting a portion of the reaction mixture onto Whatman DE81 DEAE-cellulose discs, which were washed and counted as described previously (37).

Where noted, SSB and/or primase was added to the various types of rolling circle DNA replication assays. When primase was included in the reaction mixture, CTP, UTP, and GTP were also added, each to a final concentration of 67 µM. We measured the sensitivity of newly synthesized DNA to a nuclease specific for single-stranded DNA to determine whether the lagging strand had been synthesized when the reaction mixture also contained primase and rNTPs. Each reaction mixture was brought to 0.8% SDS and heated for 10 min at 65 °C. S1 nuclease buffer (13 volumes) and 300 units of S1 nuclease were added to all samples, which were incubated for 30 min at 37 °C and then analyzed for the presence of acid-insoluble radioactivity as described above.

Highly viscous DNA products were produced when primase or primase and SSB were present during rolling circle DNA synthesis that was initiated with the assistance of the  O and P proteins. Such DNA products, if labeled with ³H, needed to be digested partially with DNase I to obtain optimal counting efficiencies. Following completion of DNA synthesis, reaction mixtures were supplemented with DNase I (0.001 unit/25 µl of reaction mixture) and incubated for an additional 5 min at 30 °C before analysis.

The efficiency of initiation of rolling circle DNA replication was determined using radioactively labeled TNC DNA as the template. DNA replication was carried out as described above using unlabeled dNTPs. Replication products were resolved from unutilized template DNA by electrophoresis in a neutral agarose gel. Following electrophoresis, the gel was dried onto Whatman DE 81 paper and autoradiographed (see below). The band containing the TNC DNA template and the band containing tailless nicked circular DNA were excised, using the autoradiograph as a guide. The remainder of each lane was cut into 8 equivalent segments. The bottom 1-cm segment had no detectable DNA product and was used to determine the background level of radioactivity (B). The individual segments of paper were counted in a liquid scintillation spectrometer. The amount of template utilized at each time point was calculated by the following formula: % utilized = 100  100{[(S  B)/T]/[(S_t = 0  B_t = 0)/T_t = 0]}, where S represents the amount of label in the TNC DNA band, B represents the amount of background label, and T represents the total amount of label, corrected for background, in the entire lane. The proportion of tailless nicked circular DNA in each lane was also determined.

DNA from restriction digests and products of rolling circle DNA replication were electrophoresed under native conditions in 1% agarose gels (13.5 cm long) in TGE buffer. Electrophoresis was carried out for 4 h at 4.25 V/cm. If a specific DNA species was to be recovered by electroelution, the agarose gel was stained briefly with TGE buffer containing 5 µg/ml ethidium bromide, and the desired DNA band was visualized under 300-nm ultraviolet light and excised. DNA was electroeluted from the agarose slice in TGE buffer in an Elutrap apparatus (Schleicher and Schuell) as detailed by the manufacturer. For autoradiography, agarose gels were dried onto DEAE-cellulose paper (Whatman DE81) in a slab gel dryer (Hoefer Scientific Instruments) and then exposed to Kodak XAR-5 x-ray film at 80 °C in the presence of a DuPont Cronex Lightning-Plus intensifying screen.

The chain lengths of replication products were determined from their electrophoretic mobilities in 30-cm alkaline agarose gels. The gels contained 30 mM NaOH, 2 mM EDTA, 0.1% SDS, and 0.4% agarose. The DNA samples were denatured by the addition of alkaline gel sample buffer (0.25 volume) and were electrophoresed at room temperature at 1.6 V/cm for 60 h. The gels were transferred immediately onto DEAE-cellulose paper, without neutralization or staining, and dried and exposed to film as described above. The sizes of the largest ssDNA molecules in a population were determined by measuring the relative mobility (R_m) at the back edge of the slowest migrating products relative to an internal standard and comparing the R_m obtained with the relative mobilities of known molecular weight standards present on the same agarose gel. The internal standard used in these experiments was pEMBL130(+) DNA that had been linearized with BamHI and end-labeled with ³²P.

DNA concentrations were measured by absorbance at 260 nm in a spectrophotometer or by a dye-binding assay, using Hoechst 33258, in a TK100 DNA fluorimeter (Hoefer Scientific Instruments) carried out according to the manufacturer's instructions. The DNA molecular weight markers and the DNA internal standard used for the alkaline agarose DNA gels were treated with calf intestine phosphatase followed by end labeling with T4 polynucleotide kinase and [-³²P]ATP (38).

RESULTS

During the initiation of bacteriophage  DNA replication, the phage-encoded O and P replication proteins participate in the assembly of the replication fork apparatus by transferring one or more molecules of DnaB helicase onto the viral chromosome. To investigate if the  O and P proteins have any detectable effects on replication forks that they help establish, we decided to compare the functional properties of such forks with those of a minimal replication fork apparatus composed of DnaB helicase and DNA pol IIIh. We chose to use TNC DNA molecules as the replication template in these studies, since Mok and Marians (39) had earlier shown their effectiveness as templates for the establishment of rolling circle DNA replication accomplished by the precisely coordinated action of the helicase activity of DnaB and the DNA synthetic activity of DNA pol IIIh. The TNC DNA template used throughout this study is a 4.0-kb nicked circular duplex DNA carrying a 758-nucleotide single-stranded DNA tail at the 5 terminus of the nicked strand. It was prepared in several steps. Initially, an 800-nucleotide ssDNA fragment was released from M13mp19oriL DNA. This was accomplished by digestion of this single-stranded circular phage DNA with PstI and SacI after first converting the unique recognition sequences for these restriction nucleases into a double-stranded form by the hybridization of a short complementary oligonucleotide. The 800-mer ssDNA fragment was isolated and hybridized to pEMBL130(+) DNA via a 42-nucleotide region at the 3 terminus of the fragment that is complementary to the polylinker sequence present on pEMBL130. The primed ssDNA circle that results was converted to a duplex form, the TNC DNA template, by replication with DNA pol IIIh in the presence of SSB.

An important early step in the establishment of rolling circle DNA synthesis on a TNC DNA template in our in vitro system is the assembly of a molecule of DnaB helicase on the 5-ssDNA tail carried by the TNC template. This ssDNA tail was purposely made long to maximize the opportunity for DnaB to interact with the TNC template. Once transferred onto the ()-strand ssDNA tail, DnaB will, in the presence of ATP, translocate along the strand in the 5  3 direction and initiate unwinding of the duplex portion of the TNC DNA template (14). This unwinding mediated by DnaB can be coupled by protein-protein interactions (40) to DNA chain elongation from the 3 terminus of the ()-strand by DNA pol IIIh, producing processive and rapid rolling circle DNA replication (Fig. 1) (39).

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A large molar excess of DnaB over template is required for optimal transfer of DnaB from solution onto ssDNA under standard replication conditions (14). Therefore, we initially explored various approaches for assembling DnaB helicase molecules on the 800-base ssDNA tail of the TNC DNA template, since the relative efficiency of this step might determine the overall efficiency of rolling circle DNA replication on the TNC DNA template. In the standard assay, DnaB is simply mixed with the DNA template in the presence of ATP at 0 °C (Fig. 1A), and DNA synthesis is initiated by the addition of DNA pol IIIh and dNTPs and warming of the reaction mixture to 30 °C. In some experiments, we included 200 µM AMP-PNP in the DnaB-binding step, since it has been reported that the binding of DnaB to ssDNA is increased when AMP-PNP is substituted for ATP (41). Although AMP-PNP is incapable of supporting the helicase activity of DnaB, it does not interfere significantly with the binding of ATP to DnaB (14, 42). Thus, once DnaB is bound to ssDNA in the presence of AMP-PNP, the addition of sufficient quantities of ATP permits helicase function by DnaB and the establishment of the elongation phase of the rolling circle replication reaction (Fig. 1A).

An alternate pathway for the transfer of DnaB onto ssDNA or TNC DNA templates involves the use of the  O and P proteins (19, 20). This multistep transfer reaction is relatively independent of DNA sequence but does require the action of the DnaK/DnaJ/GrpE molecular chaperone system. Additionally, unlike the binding of DnaB alone, the O- and P-mediated transfer reaction (Fig. 1B) is not blocked by stoichiometric coating of ssDNA by SSB (19).

In the absence of DnaB, only very modest amounts of DNA synthesis were obtained (about 50 pmol) when the TNC DNA template was incubated with DNA pol IIIh and dNTPs alone (Fig. 2). Extensive DNA synthesis was obtained, however, when DnaB helicase was included along with DNA pol IIIh in the reaction mixture (Fig. 2, squares). Maximal DNA synthesis under these conditions occurred when the reaction mixture contained approximately 60 molecules of DnaB hexamer/molecule of DNA template. This is essentially identical to the DnaB stoichiometry required for maximal strand displacement in the standard helicase assay (14). Preincubation of DnaB with the TNC DNA template for 5 min at 30 °C in the presence of AMP-PNP had little noticeable effect on the level of DnaB required to saturate the RCR assay (data not shown).

Optimization of the levels of DnaB and the P·DnaB complex in the RCR and  RCR assays, respectively.

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Significantly reduced amounts of DnaB were found to be required for saturation of the RCR assay when DnaB was transferred onto DNA with the aid of the  O and P replication initiation proteins (Fig. 2, open and closed circles). For simplicity, the RCR assay that uses the  O and P proteins to load DnaB onto the TNC template will be referred to as the  RCR assay. In such assays, DnaB was first converted into a P·DnaB complex with the addition of excess P protein before its addition to the TNC DNA. This mixture was subsequently supplemented with  O, the DnaK, DnaJ, and GrpE molecular chaperones, and DNA pol IIIh. Preincubation of DnaB with  P eliminates the possibility that free DnaB helicase binds to the TNC DNA (10) and contributes to the observed DNA synthesis. Although the reaction mixtures contained substantially fewer DnaB molecules when the  RCR assay was used, maximal DNA synthesis was found to increase 2-3-fold (Fig. 2, open and closed circles). Nevertheless, the presence of excess P·DnaB in the  RCR assay was found to be strikingly inhibitory. In contrast, excess DnaB was not harmful to the standard RCR assay (Fig. 2, squares). The presence of E. coli SSB in the  RCR assay reproducibly stimulated maximal DNA synthesis by approximately 50% (Fig. 2, closed circles).

DNA synthesis in the  RCR assay requires, in addition to the P·DnaB complex,  O, DNA pol IIIh, and the E. coli DnaJ and DnaK heat shock proteins (Table I). GrpE, a member of the DnaK molecular chaperone system, stimulates DNA synthesis in the RCR assay (Table I) but becomes absolutely essential in both this assay and the  SS replication system (20) only at lower DnaK concentrations (data not shown). These latter findings are consistent with earlier observations that GrpE is not required for initiation of  DNA replication in vitro unless DnaK concentrations are lowered to suboptimal levels (2, 4).

Table I. Protein requirements for DNA replication in the  RCR assay The  RCR assay mixtures were assembled as described under "Experimental Procedures," except that, where designated, individual components were omitted. Once assembled, all reaction mixtures were incubated for 30 min at 30 °C. The values listed are the average of duplicate samples. Component omitted DNA synthesis pmol None 2300   O 46   P·DnaB 23 DnaJ 18 DnaK 11 GrpE 1400 DNA pol IIIh 7

We examined the influence of various reaction conditions on the time course of DNA synthesis in the RCR assay. Previous studies have demonstrated that DNA pol IIIh has negligible strand displacement activity, displacing no more than 1-5 nucleotides (43) when it encounters in its path a 5 terminus of a polynucleotide chain base paired to the template strand. Not surprisingly, minimal DNA synthesis was obtained when DNA pol IIIh alone was incubated with the TNC template in the presence of dNTPs and ATP for 1 h (Fig. 3A, open circles). The inclusion of SSB in the reaction mixture, however, stimulated DNA synthesis significantly (Fig. 3A, closed circles). To determine whether DNA pol IIIh has an intrinsic strand displacement activity in the presence of SSB or is simply filling in gaps in the TNC template, the replication products produced at early time points in the presence and absence of SSB were analyzed by alkaline agarose gel electrophoresis. In the absence of SSB, DNA pol IIIh appears to label the linear strand of the TNC template by an idling reaction at the primer termini. Labeled DNA strands of 5.5 and 4.7 kb were detected, but no significant extension of these strands occurred during the 1-min incubation (Fig. 4A). When SSB is added, a modest strand displacement DNA synthesis activity is seen (Fig. 4B) that produces a fork movement rate of approximately 50 bp/s (Fig. 4C). It is apparent that the strand displacement activity of DNA pol IIIh observed in the presence of SSB is relatively feeble. DNA chains with a length of approximately 5.3 kb accumulate during the incubation, an indication that the DNA polymerase may pause for extensive periods at certain sites or possibly even terminate DNA synthesis at such sites. The 8.8-kb band observed in Fig. 4B may result from DNA polymerase-mediated end-labeling at the 3 termini of broken M13K07 helper phage DNA molecules (8.7 kb), which are present in trace amounts in the TNC preparation.

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It has been found that many preparations of DNA pol IIIh contain significant amounts of DNA helicase II (UvrD protein) (44). For this reason, we determined if the strand displacement activity observed when DNA pol IIIh and SSB were incubated with the TNC template resulted from UvrD helicase contamination of the DNA pol IIIh preparation. This was found not to be the case. The strand displacement activity obtained with DNA pol IIIh purified from an E. coli strain carrying a deletion of the uvrD gene (SK6776) was essentially identical to that displayed by DNA polymerase isolated from wild type cells (data not shown).

The effect of DnaB helicase on the time course of DNA synthesis by DNA pol IIIh on the TNC template is illustrated in Fig. 3B. If DnaB is permitted to bind to the ssDNA tail of the TNC template before the addition of DNA pol IIIh, DNA synthesis remains linear for approximately 10 min before slowing almost to a halt 30 min after initiation (Fig. 3B, open circles). In contrast, if SSB is added to the reaction mixture after DnaB has first been permitted to bind to the TNC template, DNA synthesis was much more extensive and was linear for a more prolonged period of time (Fig. 3B, closed circles). Nonetheless, SSB also demonstrated an inhibitory effect on RCR DNA synthesis when it was added simultaneously with DnaB to the TNC template (Fig. 3B, squares). This inhibition presumably reflects the capacity of SSB to bind rapidly to the ssDNA tail of the TNC template and interfere with the loading of DnaB onto the DNA chain (45).

We examined the effect of a preincubation of DnaB with the TNC template on the early DNA synthesis kinetics in the RCR assay. Preincubation of DnaB with the template in the presence of the ATP analogue AMP-PNP permitted constant DNA synthesis from the earliest time points after the initiation of DNA replication (Fig. 5, open circles). On the other hand, a striking lag in the kinetics of DNA synthesis was observed when no nucleotide was present during the DnaB preincubation with the template (Fig. 5, closed circles). We infer that transfer of DnaB onto the TNC template and its activation as a helicase is relatively slow, since preincubation of the TNC template with DnaB and AMP-PNP provided for more synchronous establishment of maximal DNA synthesis rates in the RCR assay.

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We also examined the effect on the DNA synthesis kinetics of using the  O and P proteins to transfer DnaB onto the TNC template. The kinetics of DNA synthesis in the  RCR assay, carried out in the presence and absence of SSB, is depicted in Fig. 3C. In the absence of SSB (Fig. 3C, open circles), DNA synthesis proceeds in a linear fashion for approximately 30 min after a short initial lag. Both the rate and extent of DNA synthesis in the  RCR assay are elevated if SSB is present, and the early lag is less prominent (Fig. 3C, closed circles).

Rates of Replication Fork Movement in the RCR and  RCR Assays

The foregoing results indicate that the kinetics and extent of DNA synthesis in the rolling circle DNA replication assays are dependent upon the manner by which the DnaB helicase is loaded onto the TNC template. In view of this, we examined if the mechanism used to load DnaB affected the rate of DNA chain elongation during rolling circle DNA replication. We first compared the rate of DNA chain elongation obtained when DnaB was loaded onto the TNC template with the aid of AMP-PNP (Fig. 6A) with the rate yielded when the  O and P replication proteins were used to deliver DnaB onto the template (Fig. 6B). The sizes of product DNA chains were estimated from their electrophoretic mobilities in alkaline agarose gels as compared with the mobilities of DNA chains of known length. To assess the maximal rate of DNA chain elongation, we measured the sizes of the longest DNA chains in each population of replication products and plotted these sizes against the reaction time (Fig. 6C). Analysis of this data indicated that the fork movement rate was 720-750 base pairs/s at 30 °C in both the RCR assay (Fig. 6A) and the  RCR assay (Fig. 6B). We conclude that the rate of replication fork movement is independent of the manner used to load DnaB onto the DNA.

Alkaline agarose gel electrophoretic analysis of the rates of DNA chain elongation in the RCR and  RCR assays. A, a large RCR reaction mixture (200 µl) was assembled. DnaB (22.4 pmol) was preincubated with 336 fmol of TNC DNA template in the presence of 200 µM AMP-PNP at 30 °C for 5 min. Rolling circle DNA replication was initiated by the addition of prewarmed DNA pol IIIh (1600 units), ATP, and dNTPs (containing [-³²P]dCTP) and incubation of the reaction mixture at 30 °C. After varying incubation times, portions (25 µl) of the reaction mixture were mixed with an equal volume of 0.1 M EDTA and further supplemented with an internal reference size standard (end-labeled DNA, 4.0 kb in length). The DNA products were electrophoresed in a 0.4% alkaline agarose gel as described under "Experimental Procedures." Lane 1, end-labeled TNC template; lane 2, end-labeled, HindIII-digested  DNA; lane 3, end-labeled, BamHI-digested pEMBL130(+) DNA; lanes 4-9, DNA products present 10, 20, 30, 40, 50, and 60 s, respectively, after the initiation of DNA replication; lane 10, end-labeled  DNA; Lane 11, end-labeled T7 DNA. B, a  RCR reaction mixture (400 µl) was assembled as follows. The TNC DNA template (672 fmol) was preincubated for 2 min at 30 °C in the presence of ATP, SSB (35.2 µg), and 16-fold quantities of  O,  P·DnaB complex, DnaJ, DnaK, and GrpE. Rolling circle DNA replication was initiated by the addition of a prewarmed mixture containing DNA pol IIIh (3200 units) and dNTPs (containing [-³²P]dCTP), and incubation was continued at 30 °C. At varying times after the initiation of DNA synthesis, 40-µl portions were removed and mixed with an equivalent volume of 0.1 M EDTA to terminate DNA replication. Portions of each sample (0.6 volume) were supplemented with end-labeled 4.0-kb DNA (an internal size standard) and were electrophoresed in 0.4% alkaline agarose gels. Lane 1, end-labeled, HindIII-digested  DNA; lanes 2-10, DNA products present after 10, 20, 30, 40, 50, 60, 70, 80, and 180 s, respectively, of DNA replication; lane 11, end-labeled  DNA. C, determination of the rates of replication fork movement. The lengths of the longest replication products at each time point were determined for several different replication time courses (such as those pictured in panels A and B) and plotted against the reaction time. A, , RCR assay described for panel A, which included a preincubation of DnaB with the TNC template in the presence of AMP-PNP; , RCR replication as described for panel A, except that a lower concentration of DnaB was used (1 pmol of DnaB/25 µl of reaction volume), and DNA chain elongation was carried out in the presence of SSB (2.2 µg/25 µl of reaction volume) (gel not shown); ,  RCR assay as described under "Experimental Procedures" (gel not shown); ,  RCR assay described for panel B, in which SSB (2.2 µg/25 µl of reaction volume) was present during DNA chain elongation.

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We also examined the effect of SSB on the rate of replication fork movement in both the RCR assay and the  RCR assay. Our results indicate that SSB has no discernible effect on the movement rate of replication forks in either type of assay (Fig. 6C). Nevertheless, we did find with the RCR assay that the addition of SSB to the TNC template before the addition of DnaB helicase strongly inhibited DNA synthesis. Furthermore, replication forks assembled in this manner moved at the slow rate (40-50 bp/s) observed when DNA pol IIIh and SSB were present but DnaB was absent (Fig. 4B and data not shown). This result was anticipated, since complete coating of the single-stranded DNA tail with SSB would be expected to block loading of the DnaB helicase onto the TNC template (19, 45).

DNA pol IIIh, the replicative DNA polymerase used in the RCR and  RCR assays, is a complex enzyme consisting of at least 10 subunits (30, 46, 47). As mentioned earlier, it has been reported that many preparations of DNA pol IIIh contain significant levels of DNA helicase II, the product of the E. coli uvrD gene (44). Western blot analysis of our DNA pol IIIh preparation with antibody specific for UvrD confirmed the presence of DNA helicase II and indicated further that approximately 20-40 ng of UvrD was being added along with DNA polymerase IIIh to individual RCR assays. It was of interest, therefore, to determine if the presence of UvrD helicase in the RCR assays influenced the results reported here. We purified DNA pol IIIh from an E. coli strain carrying a deletion of the uvrD gene and assessed the rates of replication fork movement when the UvrD-free DNA pol IIIh served as the replicative DNA polymerase in the RCR and  RCR assays. We found that removal of UvrD from the DNA pol IIIh preparation had no detectable effect on fork movement rates (data not shown). However, the two DNA pol IIIh preparations did behave differently in one respect. Preincubation of DnaB with the TNC template in the presence of AMP-PNP resulted in a 2- or 3-fold stimulation of DNA synthesis in the standard RCR assay (30-min incubation) when the DNA pol IIIh was from uvrD⁺ cells (data not shown; also see Fig. 5). In contrast, the preincubation of DnaB with the TNC template and AMP-PNP did not stimulate DNA synthesis when UvrD DNA pol IIIh was used (Fig. 7; compare closed and open circle points with no added UvrD). Similarly, the absence of UvrD in the DNA pol IIIh preparation was associated with a nearly complete elimination of the lag in early DNA synthesis kinetics previously seen (Fig. 5) when this AMP-PNP preincubation was omitted (data not shown). These results suggest that loading of the DnaB helicase onto the TNC template may be inhibited by the presence of UvrD in the DNA pol IIIh preparation. To examine this possibility, we determined the effect of adding purified UvrD protein to rolling circle replication reactions carried out by UvrD DNA pol IIIh (Fig. 7). In control experiments, UvrD stimulated DNA synthesis only slightly in the absence of DnaB (Fig. 7, squares). Added UvrD, however, had a striking inhibitory effect on DnaB-dependent RCR DNA synthesis (Fig. 7, open circles). This inhibitory effect of UvrD was partially ameliorated if DnaB was allowed to bind to the tailed DNA template, during a preincubation with AMP-PNP, before the addition of UvrD to the RCR reaction (Fig. 7, closed circles). Thus, the severalfold stimulatory effect noted above of the AMP-PNP preincubation on RCR DNA synthesis would be explained if the DNA pol IIIh (from uvrD⁺ cells) added to the reaction mixture contained between 20 and 100 ng of UvrD protein.

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Previous studies have demonstrated that primase, the product of the E. coli dnaG gene, primes synthesis of all DNA strands during replication of the chromosomes of both E. coli and phage  (48). Synthesis of RNA primers is highly dependent on transient interactions of primase with DnaB helicase bound to the template strand (15, 45, 49, 50). Accordingly, it was of interest to examine the effect of primase on DNA synthesis levels and fork movement rates in the RCR and  RCR assays. As expected, in the absence of DnaB, primase had no effect on the levels of DNA synthesis obtained in the RCR and  RCR assays, whether or not SSB was present (Table II, lines 1-4 and 9-12). We conclude that primase does not affect the strand displacement activity of DNA pol IIIh. SSB stimulated DNA synthesis in the RCR assay when DnaB was absent (Table II, lines 1 and 3), as noted earlier (Fig. 4). On the other hand, in the case of the  RCR assay, SSB failed to stimulate DNA synthesis when DnaB was absent (Table II, lines 9 and 11). Additional studies indicated that the presence of  O was responsible for blocking the SSB-mediated stimulation of strand displacement DNA synthesis in this situation (data not shown).

Table II. Effects of SSB and primase on the level of rolling circle DNA replication All RCR assays that contained DnaB included a preincubation of DnaB with the TNC template DNA in the presence of AMP-PNP as described under "Experimental Procedures." Where indicated, primase (100 ng) was also included in the preincubation mixture. SSB (2.2 µg) was added, where specified, to each mixture just before the initiation of DNA synthesis. For the  RCR assays, primase (100 ng) and/or SSB (2.2 µg) was added, where indicated, along with the other required proteins and mixed with the TNC DNA template and incubated for 5 min at 30 °C in the absence of ATP. DNA synthesis was initiated by the addition of ATP. All reaction mixtures also contained rNTPs (67 µM each), which were added just before the initiation of DNA synthesis. All reaction mixtures were incubated for 15 min at 30 °C and then processed to determine the level of DNA synthesis. The values listed represent the average of duplicate samples. Primase SSB DNA synthesis Average lengtha pmol kb RCR assay   1. Without DnaB     60 2   2. Without DnaB +   50 2   3. Without DnaB   + 580 20   4. Without DnaB + + 430 15   5. With DnaB     1100 37   6. With DnaB +   4200 71b   7. With DnaB   + 3900 130   8. With DnaB + + 4600 78b   RCR assay   9. Without  P, without DnaB     40 1   10. Without  P, without DnaB +   50 2   11. Without  P, without DnaB   + 30 1   12. Without  P, without DnaB + + 30 1   13. With  P·DnaB     3300 110   14. With  P·DnaB +   5100 86b   15. With  P·DnaB   + 4800 160   16. With  P·DnaB + + 11,100c 190b