Proc. Natl. Acad. Sci. USA Vol. 80, pp. 5817-5821, October 1983 Biochemistry Purified dnaA protein in initiation of replication at the Escherichia coli chromosomal origin of replication (oriC /site-specifie DNA binding/RNA polymerase/DNA gyrase) RoBERT S. FULLER AND ARTHUR KORNBERG Department of Biochemistry, Stanford University School of Medicine, Stanford, California 94305 Contributed by Arthur Kornberg, May 26, 1983 ABSTRACT Soluble protein fractions from Escherichia coli dnaA* cells but not dnaA temperature-sensitive cells replicate plasmids containing the E. coli chromosomal origin of replication (oriC). Complementation of these mutant fractions provided an assay for dnaA protein activity in initiation of replication at oriC. From a strain (constructed in vitro) that overproduces the dnaA protein more than 200-fold, the 52,000-dalton polypeptide was pu- rified to near homogeneity. Although the protein tends to aggre- gate, monomer-sized protein purified by high-performance liquid chromatography is fully active for replication. It binds specifically and tightly to oriC in a supercoiled plasmid as judged by a Mil- lipore filter-binding assay and by protection of the unique HindIII site within the oriC sequence. In the oriC replication reaction, dnaA protein acts at an early step preceding DNA synthesis. The dnaA gene was identified by conditional lethal mutations near 82 min (on the revised Escherichia coli map) that were defective at an elevated temperature in initiation of a cycle of chromosome replication (1, 2). The dnaA gene product is thought to act early in initiation, at about the same time as RNA poly- merase (3). Several second-site suppressors of dnaA mutations that map within rpoB suggest a direct interaction between RNA polymerase and the dnaA gene product (4, 5). Besides the E. coli chromosomal origin of replication (oriC), only the plasmid pSC101 requires dnaA function for sustained replication in vivo (6, 7). This replicon specificity, early action, and functional in- teraction with RNA polymerase suggest that dnaA action is tar- geted directly to oniC. The dnaA gene has been cloned in A transducing phages by exploiting its close linkage to tna (8). The reported molecular weight of the dnaA polypeptide has ranged from 48,000 to 54,000 (8-11); a molecular weight of 52,574 has been calculated from the complete sequence of the gene (12). Replication of oriC-containing plasmids in vitro provides a novel way to study the dnaA product (13). Replication of oriC plasmids in this system resembles authentic in vivo replication by several criteria, including absolute dependence on dnaA function (13, 14). This system has provided an assay for puri- fication of dnaA protein from strains constructed in vitro that overproduce the protein more than 200-fold. The purified pro- tein binds oriC and participates at an early stage in initiating bidirectional replication from oriC. MATERIALS AND METHODS Strains and Phages. Strains were: WM433 (dnaA204) from W. Messer (13), N4830 (cI857) from H. Echols (15), and K37 (Hfr) from J. M. Kaguni (16); phages M130riC26 and M13oriC26A221] were from J. M. Kaguni (16). The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked “advertise- ment” in accordance with 18 U.S.C. §1734 solely to indicate this fact. 5817 Reagents and Buffers. Hepes, polyvinyl! alcohol (type IJ), creatine kinase (type I), creatine phosphate, ribonucleoside tri- phosphates, and uridine were from Sigma; dNTPs were from P-L Biochemicals. [7H]Thymidine triphosphate (30-40 Ci/mmol) and [3H]thymidine (75 Ci/mmol) were from New England Nu- clear (1 Ci = 3.7 x 10!° Bq). Buffer C is 25 mM Hepes-KOH, pH 7.6/0.1 mM EDTA/2 mM dithiothreitol /20% sucrose; buffer C’ differs only in that Hepes‘KOH is 50 mM; buffer D is buffer C' with addition of Mg(OAc), to 10 mM, KCI to 0.1 M, and (NH4)2SO4 to 0.2 M. Enzymes and Proteins. T4 lysozyme was prepared as cited (13); EcoRI was a gift of P. Modrich; HindIII was from New England BioLabs. Protein was assayed by the method of Brad- ford, with bovine serum albumin (Miles) as a standard (17). Preparation of DNAs. Unlabeled phage M 130riC 26 repli- cative form (RF) I DNA was prepared as described (13). For °H- labeled RF I, 1.5-liter cultures of strain K37 were grown in M9 medium (18) containing 0.1% Casamino acids (Difco), 0.2% glu- cose, and thiamine (1 xg/ml). At an ODsgs of 0.3, uridine was added to a final concentration of 200 g/ml along with 5 mCi of [*H]thymidine. At an ODsos of 0.4, phage M130riC26 or M13oriC26A221 was added at a multiplicity of infection of 80; the cells were harvested after 2 hr, and RF I was purified by the same procedure used for unlabeled DNA; specific activities were 96,000 cpm/jg. To prepare DNAs linearized by EcoRI or HindIII, 5 yg of 9H-labeled M13oriC26 RF I or 9H-labeled M130riC26A221 RF I in 50 yl of medium salt buffer (19) was treated for 30 min at 37°C with 10 units of restriction enzyme. Assay of Replication Activity of dnaA Protein. Comple- mentation of oriC replication in fraction II from a dnaA mutant strain WM433, prepared as previously described (13), was in a volume of 25 yl containing Hepes‘KOH (pH 7.6), 40 mM; GTP, CTP, and UTP, each at 0.5 mM; ATP, 2 mM; dGTP, dCTP, TTP CH at 48 cpm/pmol), and dATP, each at 100 4M; magnesium acetate, 11 mM; polyvinyl alcohol, 7% (wt/vol) creatine phos- phate, 40 mM; creatine kinase, 100 ug/ml; WM433 fraction IT protein, 300 ug; and M13oriC26 RF I, 200 ng (600 pmol as nu- cleotide). Components, except for creatine kinase and DNA, were assembled on ice and centrifuged for 10 sec in an Ep- pendorf microfuge. Creatine kinase, template DNA, and the dnaA protein fraction to be assayed were added to the super- natant. After 20 min at 30°C, the extent of DNA synthesis was determined as described (13). One unit of dnaA protein activity (in the linear range of 300-800 pmol of dNMP incorporation) is 1 pmol of dNMP incorporated per min. Assay of oriC Binding Activity of dnaA Protein. Reaction mixtures (25 yl), assembled on ice, contained Hepes*KOH (pH 7.6), 40 mM; KCl, 150 mM; Mg(OAc),, 10 mM; dithiothreitol, 2 mM; bovine serum albumin, 100 g/ml; and *H-labeled DNA, 200 ng. After addition of dnaA protein, reactions were incu- Abbreviation: RF I, replicative form I DNA. 5818 Biochemistry: Fuller and Kornberg bated at 30°C and then spotted onto 2.4-cm Millipore HA filters (pore size, 0.45 ym). Filters, prepared by boiling in four changes of quartz-distilled water and stored at 4°C in water, were soaked in wash buffer (reaction buffer without bovine serum albumin and DNA) at room temperature for 30 min before use. Filtra- tion by gentle suction was followed by washing with 0.5 ml of wash buffer at 30°C and drying under an infrared lamp. Ra- dioactivity was determined by liquid scintillation counting. Maximal efficiency of retention of the complex was 80-90%; this correction has not been applied to the results. RESULTS Purification of dnaA Protein. Table 1 summarizes the pro- cedure. The protein was overproduced by inserting the dnaA gene into vector pAD329 (20) to generate plasmid pBF 110 (un- published data; this plasmid will be furnished on request) (Fig. 1). Amplification by 200-fold of the dnaA protein level resulted from enhanced transcription of the dnaA gene from the A phage Pu promoter induced by temperature inactivation of the prod- uct of the cl857 gene in strain N4830. A heat-produced lysate in 20 mM spermidine-HCl (21) could be clarified by low-speed centrifugation and yielded the same amount of dnaA protein activity with only half as much protein as in a freeze-thaw lysate (13). Chromatography of the am- monium sulfate fraction (fraction II) on Bio-Rex 70 and elution with a salt gradient separated activity into two broad peaks; the second contained more than half of the starting activity and was 50-80% pure as judged by NaDodSO,/polyacrylamide gels stained with Coomassie blue (unpublished data). When activity that had been eluted in the high-salt peak was rechromato- graphed on Bio-Rex 70, 80% was eluted again with high salt. When dnaA protein eluted by high salt (0.8-0.9 M KC}) from Bio-Rex 70 was chromatographed by HPLC, two peaks of rep- lication activity were observed (Fig. 2), each corresponding to the abundance of the 52,000-dalton band in NaDodSO,/poly- acrylamide gel electrophoresis. The excluded (or just slightly included) protein in the first peak is presumably a multimer or aggregate; the second peak (eluted just after the position of bo- vine serum albumin) corresponds to monomeric dnaA protein. Both forms of dnaA protein were active in the replication (com- plementation) assay, although interconversion of these forms was not observed by HPLC after exposure to pH values of 6.0- 7.5 and salt levels of 0.2-1.0 M KCI. Monomeric dnaA protein, about 95% pure as judged by the gel pattern, had a 2.5-fold higher specific activity in the replication assay than did the high molecular weight form. One circle is replicated per 15 mono- meric dnaA protein molecules added to the replication assay. Early Action of dnaA Protein in Initiation. In an oriC rep- lication system partially reconstituted from purified compo- nents (unpublished data), dnaA protein was observed to act early (Fig. 3). As in the crude dnaA complementation assay (14), there was a lag of 3-5 min before DNA synthesis began. Prior in- cubation (“preincubation”) of all components in the absence of dNTPs eliminated this lag; DNA synthesis began immediately upon addition of dNTPs. When either DNA gyrase (E. coli to- poisomerase II) or RNA polymerase was added only after the preincubation (along with dNTPs), there was no reduction in lag time (unpublished data), implying that both are required for the earliest events. When dnaA protein was omitted from the preincubation, DNA synthesis began with an abbreviated but significant lag (about 1.5 min) after its addition (Fig. 3). Thus, dnaA protein participates in an early stage of the reaction but perhaps after the action of gyrase and RNA polymerase. Specific Binding of dnaA Protein to the oriC Sequence. Par- tially purified dnaA protein specifically retained restriction Proc. Natl. Acad. Sci. USA 80 (1983) Table 1. Purification of dnaA protein Specific . Activity, activity, Fraction Protein, units units/mg Yield, No. Description mg x 10°5 x 10-8 % I Cleared lysate 9,120 TI Ammonium 3,300 210 6.2 (100)* sulfate Tl Bio-Rex 70+ A 55 63 116 30 B 25 105 415 | 98 Cc. 8.0 37 458 18 IV HPLC? A§ 2.3 13 590 7 758 BS 11 15 1,400 7 Strain N4830 (pBF110) was grown in 200 liters of L broth (18) con- taining thymine, 25 ug/ml; glucose, 0.2%; and ampicillin, 25 ug/ml, at 30°C to an Agog of 0.8, shifted to 39°C, and harvested by centrifu- gation after 1.5 hr at an ODso, of 2.4. The cell paste was resuspended in buffer C containing 250 mM KCI to an ODsp5 of 400, frozen in liquid nitrogen, and stored at —80°C. Thawed cell suspension (408 g) was di- luted 2-fold with buffer C containing 250 mM KC]; brought to 20 mM in spermidine HCl, 200 »g/ml in egg white lysozyme, and 0.1 ug/ml in T4 lysozyme; left at 0°C for 30 min; and lysed in 250-ml batches in centrifuge bottles placed at 37°C for 4 min and inverted each min. Ly- sates were chilled to 0°C and centrifuged 70 min at 14,000 rpm in a Beckman JA14 rotor, and the supernatant was collected (570 ml, frac- tion I). (NH,)2SO, (160 g) was added to fraction I, and the suspension was stirred at 0°C for 30 min and centrifuged as above. The pellets were resuspended in 34 ml of buffer C’ to a final volume of 50 ml (fraction 11), dialyzed for 2 hr against 1 liter of buffer C’, and diluted with 215 ml of buffer C‘ to a conductivity equivalent to that of buffer C’ con- taining 50 mM KC). Dialyzed fraction II was loaded at 4°C onto a 250- mi Bio-Rex 70 (100-200 mesh) column equilibrated with buffer C’ con- taining 50 mM KCI by first stirring with 80 ml of the packed resin and then pouring this slurry on the column containing the remaining resin. The column was washed with 7 column volumes of buffer C’ containing 50 mM KCI, and activity was eluted with a gradient (2 liters) of 50 mM to 1 M KCl in buffer C in two broad peaks between 0.3 and 0.5 M KC] and 0.6 and 0.9 M KCl. Fractions with comparable specific activity were pooled (A, 0.40.5 M KCI; B, 0.5--0.8 M KCI; C, 0.8-0.9 M KC}) and precipitated with the addition of 0.35 g of (NH,)2SO, per ml. Of the dis- solved precipitate of pool C, 1.4 ml (18%) was concentrated 2-fold by vacuum dialysis (25,000-dalton-cutoff collodion bag, Schleicher & Schuell) against buffer D and chromatographed (HPLC) at 0°C at a pressure of 500 psi (1 psi = 6.89 kPa) and a flow rate of 0.6 ml/min by three successive injections on a TSK 3000 SW column (Altex, M, ex- clusion of 200,000—400,000) equilibrated with the buffer D; 0.3-ml frac- tions were collected. * Yield and purification are based on fraction II because activity could not be reliably measured in fraction I. + A,B, and C are the pools of Bio-Rex fractions after concentration with ammonium sulfate. +Only 18% of fraction IIIC was carried through to fraction IV; the val- ues are corrected for this by a factor of 5.6. $A includes HPLC fractions 5 and 6; B includes HPLC fractions 15-18. Yield in fractions IVA and B combined, relative to fraction HIC, was 15%. fragments containing oriC in a nitrocellulose filter binding as- say (25). However, dependence on DNA gyrase (13) and rel- ative inactivity of linearized oriC template (unpublished data) imply that the optimal template for in vitro replication is su- percoiled oriC plasmid DNA. Binding of dnaA protein to su- percoiled M130riC26, an M13-oriC chimera that can utilize oriC both in vivo and in vitro (13, 14, 16), was measured by Millipore filter binding (26). M13oriC26A221, having a small deletion that removes the oriC sequence, (16) served as a con- trol. dnaA protein retained both *H-labeled RF Is, and the ti- tration curve of dnaA protein was sigmoidal in each case (Fig. 4A). A 3-fold preference for M13oriC26 RF I (Fig. 4A) was ac- centuated to greater than 30-fold by addition of heterologous, Biochemistry: Fuller and Kornberg RH BR RP (X/S) P P R ‘ i Lu bedh a ' i ppv ——P- — ——— ! dnaA galK ori bla Fic. 1. Organization of the dnaA plasmid pBF110. B, BamHI; R, EcoRI; H, HindIll; P, Pvu I; (X/S), junction of BamHI-Xhe I fragment containing dnaA [the HindIII-Xho I dnaA fragment from pBF101 (13) (am) converted to a BamHI-Xho I fragment with addition of BamHI linkers} to Sal I-BamHI vector fragment. The vector pAD329 is iden- tical to pMA22 (20) except that it lacks the Bgi Il fragment containing the phage A cll gene. Arrows refer to the directions of genes and tran- scripts. unlabeled competitor DNA (Fig. 4B). These results imply that dnaA protein recognizes a site present in M13o0riC26 but ab- sent from M130riC26A221, probably within the minimal oriC sequence. Binding activity specific for oriC coincided with both the abundance and replication activity of dnaA protein in the HPLC column fractions (Fig. 2). The ratios of oriC binding to replication activities were similar in the excluded and monomer peaks. Characteristics of the dnaA Protein—oriC’ Complex. For- mation of a dnaA protein-oriC complex was complete in less than 30 sec. When complex formed with 660 fmol of mono- meric dnaA protein (27 nM) and 63 fmol of *H-labeled oriC plasmid (2.5 nM; DNA was at saturation) was challenged with excess unlabeled oriC plasmid (250 fmol), the amount of [°H]- DNA retained (16 fmol) was reduced by 45% in 60 min, indi- cating considerable stability of the complex. Complex forma- A ad M an (4,5, 68,7, 8,10,12,14 15, 16,17 ,18,19, 20,22, Protein, mg/ml (Oo) 3 5 7 9 11 #13 «15 «+17 Fractions Proc. Natl. Acad. Sci. USA 80 (1983) 5819 tion was relatively salt resistant; in 300 mM KCl, binding was reduced by about 50% compared to standard conditions (150 mM KCl). When 3H-labeled M130riC26 was titrated to satu- ration in the presence of constant levels of dnaA protein, half- saturation was achieved with a concentration of free plasmid molecules of 0.3 nM at 27 nM dnaA protein (monomer) and with 0.6 nM free plasmid at 53 nM dnaA protein. At saturation, the ratio of dnaA monomers to oriC molecules was 50:1 and 20:1 for the two dnaA protein concentrations. Influence of Superhelicity on Binding. When either 7H-la- beled M130riC26 RF I or °H-labeled M130riC26A221 RF I was linearized by EcoRI cleavage ata site distant from oriC (16), retention of DNA was reduced significantly compared to the corresponding supercoiled DNA, yet the preference for oriC26 versus oriC26A221 was maintained (Fig. 4A). This suggests that dnaA protein binding may be accompanied by the bending or partial unwinding of the DNA favored in negatively super- coiled DNA. Protection of the HindIII Site Within oriC by dnaA Protein. M136nC26 contains single sites for cleavage by EcoRI and HindIII. The EcoRI site is within sequences derived from phage G4, whereas the HindIII site is within the highly conserved, minimal oriC sequence (27, 28). Comparison of binding to M13oriC26 and M13o0riC26A221 DNAs (Fig. 4) indicated that adnaA protein binding site or sites lay within or overlapped the region lost in the deletion A221. The protection afforded by dnaA protein binding. against HindIII cleavage directly dem- onstrates oriC specificity. Cleavage by HindHI but not by EcoRI arkers Fic. 2. Purification of dnaA protein by HPLC. (A) Bio-Rex pool C (LOAD lane, 10 1) or HPLC col- umn fractions (lanes 4—22, 100 #1) were precipitated at 0°C with. 8% trichloroacetic acid and loaded on a NaDodSO,/polyacrylamide gel (4% stacking and 12.5% body gel) (22). M, markers (shown X 10°“) were phosphorylase. b (93,000); bovine serum albumin (67,000); @X174 capsid proteins F (46,300), H (35,800), and G (19,000); and carbonic anhydrase (29,000). (B) Assays for replication and binding activities of dnaA protein. Binding assays were incubated at 30°C for 2 min in 25 ul containing 1 zg of CoE] DNA and 0.2 (a, M13 oriC26; a, M13 oriC264221) Replication activity, u/ml x 107 (o) DNA binding activity, u/ml x 1077 yg of either °H-labeled M130riC26 or *H-labeled M13o0riC26A221 RF I. One unit (u) of DNA binding activity corresponds to retention of 1 fmol of. eriC plasmid under the conditions of the assay. 5820 Biochemistry: Fuller and Kornberg 1,200 S E a a 800 an o x= < a 400