ATP activates dnaA protein in initiating replication of plasmids bearing the origin of the E. coli chromosome

Cell, Vol. 50, 259-265,

July 17, 1967, Copyright

0 1967 by Cell Press

ATP Activates dnaA Protein in Initiating Replication of Plasmids Bearing the Origin of the E. coli Chromosome Kaxuhisa Sekimizu’t David Bramhill,‘* and Arthur Kornberg’ * Department of Biochemistry Stanford University School of Medicine Stanford, California 94305

Summary ATP is bound to dnaA protein with high affinity (KI, = 0.03 PM) and hydrolyzed slowly to ADP in the presence of DNA. ADP Is also bound tightly to dnaA protein and exchanges with ATP very slowly. The ATP form is active in replication; the ADP form is not. A unique conformation of oriC, formed In an early initiation stage, depends on dnaA protein being in the ATP form. The subsequent entry of dnaB protein to form a prepriming complex also requires ATP binding and is blocked by bound ADP Inasmuch as hydrolysis of ATP is far slower than these initiation reactions and since the poorly hydrolyzable analogue ATprS can replace ATP, the ATP function appears to be allosteric. The extraordinary affinity of ATP for dnaA protein, its slow hydrolysis to ADP, the profound inhibition of dnaA functions by ADP, and the very slow exchange of ADP all point to a possible regulatory role for these nucleotides in the cell cycle. Introduction The cycle of chromosomal replication in E. coli starts at a unique, 245 bp sequence (oriC) and is regulated at the stage of initiation (Leonard and Helmstetter, 1988). The process has become accessible to biochemical analysis by the engineering of an oriC plasmid (Yasuda and Hirota, 1977) and the discovery of an enzyme system that sustains its replication (Fuller et al., 1981). dnaA protein, essential for initiation of replication in vivo (Hirota et al., 1970), also plays a crucial role in both crude (Fuller and Kornberg, 1983) and purified enzyme systems (Kaguni and Kornberg, 1984; Ogawa et al., 1985; van der Ende et al., 1985). Binding of the protein to four 9-mers (“dnaA boxes”) within the oriC sequence is an essential step for forming the first stage initiation complex that readies the plasmid for priming of DNA synthesis (Fuller et al., 1984). Beyond its specific binding to a dnaA box, little is known about the functional features of dnaA protein. In this study, we describe the tight binding of ATP and ADP to dnaA protein with crucial effects on its role in initiation of replication.

t Present address: Faculty of Pharmaceutical Sciences, University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113, Japan. $ Present address: Department of Biological Sciences, Stanford University, Stanford, California 94305.

Results dnaA Protein Has a High Affinity for ATP In the absence of ATP the replication activity of dnaA protein is unstable under conditions used for assay of dnaA protein. At 38OC, more than 50% of the replication activity was lost within 2 min and more than 95% in 10 min. This inactivation was prevented by ATP at a concentration of less than 1 PM (data not shown). Based on binding of [a-=P]ATP to dnaA protein on a nitrocellulose membrane, a Kc value of 0.03 f 0.01 PM was determined (Figure 1). The number of ATP binding sites per dnaA protein moiecule was calculated to be 0.48. In attempts to account for this lack of stoichiometry, no evidence was found for nucleotides remaining bound to dnaA protein during the course of purification (Fuller and Kornberg, 1983), nor did treatment of dnaA protein, under conditions known to remove ATP, increase the subsequent extent of ATP binding (data not shown). ATP Bound to dnaA Protein Is Hydrolyzed Slowly Hydrolysis of tightly bound ATP was 50% complete in about 15 min (Figure 2); the ADP product remained tightly bound to the protein. The ATPase activity was stimulated by DNA (Table 1) without any apparent specificity for the oriC sequence; supercoiled DNAs with the oriC sequence (pCM959, M13oriC28, M13oriC2LB5, and M13mpRE85) or without it (M13AE101, pBR322, ColEl, and @X174) were effective. Nucleotide Specificity for Binding to dnaA Protein Using competition with ATP as a measure, ADP and ATPyS bound dnaA protein about as effectively as did ATP itself (Table 2). CTP dATP A(pkA, and AMPPNP were also competitive; in view of its tight binding, contamination by ATP must be considered in appraising the specificity for weakly competitive nucleotide preparations. A 1% contamination of GTP by ATP could account for its competition. Adenosine, AMP, dGTP, dTTP and AMPPCP all failed to compete with ATP Exchange of Nucleotide Bound to dnaA Protein Is very Slow The Ko value for binding of ADP to dnaA protein, based on retention on a nitrocellulose membrane, was 0.1 NM; the number of binding sites was 0.53 per dnaA protein molecule, which is close to that for ATP Dissociation of [14C]ADP from dnaA protein in the presence of 10 PM [a-%P]ATP proceeded only half way in about 30 min (Figure 3). Similar results were obtained in the exchange of bound ATP with free ATP These very slow exchanges were not increased by any of the proteins or the oriC DNA that participate in subsequent stages of oriC plasmid replication, nor was there any stimulation by cruder soluble protein fractions.

Cd 260

Table 1. Influence of DNA on Hydrolysis Bound to dnaA Protein Supercoiled

0

0.5

DNA

of ATP Tightly

ATP Hydrolysis

None

0.10

pCM959 M 13oriC26 M 13ofiC2LB5 Ml 3mpRE85

0.88 0.98 0.98

M13AElOl pBR322 ColEl @X174

0.54 0.55 0.56 1.54

(pmol)

0.91

Hydrolysis of ATP bound to dnaA protein (6.0 pmol) was measured as in Figure 2, except with various DNAs (0.5 ug). The amounts of ADP-dnaA protein complex after a 15 min incubation were determined and presented as the average of duplicate assays (data range is 8%).

2.0

ATP (PM) Figure

1. Binding

of ATP to dnaA

Protein

dnaA protein (2.4 pmol) was incubated cpmlpmol) as described in Experimental

with [@P]ATP Procedures.

(3 x

lo4

The ATP, but Not the ADP, Complex of dnaA Protein Is Active in Replication Replication activities of the ATP and ADP complexes of dnaA protein were compared at the high concentration of ATP (2 mM) needed in the overall replication reaction after the dnaA protein had been exposed to 1 uM ATP or ADP at 0% for 15 min (Figure 4A). The ATP, but not the ADP complex was active. Inasmuch as the prior incubation of dnaA protein (15 min at 0%) without nucleotides did not decrease its activity, the effect of ADP cannot be attributed to a failure to protect dnaA protein from inactivation. In fact, exposure of dnaA protein to 1 uM ADP protected it from inactivation at 38% as did ATP (data not shown). The tight binding of ATP after the slow exchange with ADP (Figure 3) directly demonstrated the retention of dnaA pro-

tein function, as did the capacity of the exchanged form subsequently to support oriC plasmid replication. In the foregoing experiments, RNA polymerase was omitted from the oriC replication system reconstituted with pure proteins (van der Ende et al., 1985). When this system contains HU protein in an amount sufficient to coat much of the plasmid, dependence on RNA polymerase becomes nearly absolute (Ogawa et al., 1985). Under these conditions (Figure 48) the properties of the ADP and ATP forms of dnaA protein are like those observed in the system with low HU levels and lacking RNA polymerase. In addition, the ADP form is inactive in the crude protein system (fraction II), which was originally developed for assay of the replication activity of dnaA protein (Fuller et al., 1981). The failure of the ADP-dnaA protein complex to

Table 2. Nucleotide

Competitor

I

I

I

0

10

20

0

Figure

2. Time

Course

30

40 MINUTES

of Hydrolysis

50

60

of ATP Bound

70

80

to dnaA Protein

dnaA protein (6.0 pmol) was first incubated at 0°C for 15 min with 1.5 uM [&P]ATP (4 x 10s cpmlpmol) as in Figure 1, except that Mg2+ was 2.5 mM. Incubation at 38“C with 0.2 pg of pCM959 DNA followed. Samples were filtered on membranes and extracted with 400 VI of 1 M HCOOH. Samples (0.5 PI) were spotted on thin layer PEI-cellulose plates. Chromatography was with 1 M HCOOH, 0.4 M LiCI. Unlabeled standards were located by UV absorption.

Nucleotide

Specificity

of Binding

to dnaA Protein

[a-32P]ATP-dnaA (% of Control)

ATP= GTP CTP UTP

2 67 4 24

dATP dGTP dCTP dTTP

8 107 16 103

ADP AMP Adenosine

2 104 102

ATPyS AMPPNP AMPPCP

3 11 91

APPPPA

6

Protein

Purified dnaA protein (4.6 pmol) was incubated with 1 uM [03sP]ATP and 50 uM competitor nucleotide at 0°C for 15 min. The amounts of ATP-dnaA protein complex were measured by filtration through Millipore membranes. Average values of duplicates are presented. Without a competitor, 2.2 pmol of ATP-dnaA protein complex was retained on the membrane. Yfnlabeled ATP; this value agrees with that expected for competition by dilution.

ATP Activates 261

E. coli dnaA Protein

I

I

I

I

I

_ 0.05 I 0

Figure

3. Exchange

I

1

20

40

Reaction

I 60 MINUTES

I

I

80

100

Figure

of ATP with ADP Bound to dnaA Protein

Both the ATP Form and the ADP Form of dnaA Protein Bind to oriC Binding to oriC of the non-nucleotide form of dnaA protein was demonstrated by retention of [32P)oriC DNA on Millipore membranes (Fuller and Kornberg, 1983). Similar DNA binding was observed with both the ATP complex and the ADP complex of dnaA protein (Figure 5). The binding of supercoiled DNA by the two forms was also indistinguishable (data not shown). Salt (0.2 M KCI) or com-

I I POLYMERASE

40

60

80

0 MINUTES

20

40

5

Protein

60

Figure 4. Replication Activity of ATP or ADP Forms of dnaA Protein in the Solo Primase System or RNA Polymerase-Dependent System dnaA protein (0.92 pmol) was incubated with 1 PM ATR 1 nM ADP, or no nucleotide (ATPdnaA complex was assumed to be formed during replication) at 0% for 15 min. Replication activity was measured by the nonstaged reaction reconstituted with purified proteins (Cgawa et al., 1985). The reaction without dnaA protein provides the background value.

ATP

20

of oriC DNA-dnaA

2

The ADP Form of dnaA Protein Palls in Strand Opening Following binding of the dnaA protein to oriC, the next identified stage of the initiation reaction is a dnaA-dependent strand opening within oriC. This can be detected by

t

0

0.5 1 (pmol)

petitor DNA (40 ug of calf thymus DNA) showed no preferential effects on the binding by the ATP form as compared with the ADP form of dnaA protein. Specific binding of dnaA protein to the dnaA boxes in oriC had been observed by footprinting analysis in the absence of ATP (Fuller et al., 1984). With ATP or ADP complexed to dnaA protein, the protection of supercoiled template against digestion by DNAase I by the method of Gralla (1985) showed no major differences (data not shown). Thus, failure of the ADP form of dnaA protein must occur at a stage subsequent to the initial binding.

release some activity over an extended period of time (Figure 4) may be conditioned by the following: by the more rapid hydrolysis of ATP on dnaA protein in the presence of DNA (Figure 2) relative to its exchange, by the nonlinear dependence of dnaA protein binding to oriC DNA (see Figure 5) and by other differences in the reaction conditions.

I RNA

Assay

PROTEIN

Various amounts of dnaA protein were incubated with 1 uM ATP, 1 PM ADR or no nucleotide at 0% for 15 min in 85 ul of the same buffer as in Figure 3 (see Experimental Procedures). Incubation (4 min at 38%) was with 5.5 fmol(35.000 cpm) of 3’ end-labeled [32P]Smal-Xhol oriC fragment (464 bp) of pCM959. Samples were filtered through membranes (Millipore, HAWP), and radioactivity retained was counted.

dnaA protein (1.9 pmol) was incubated with 1 PM [‘%]ADP (1100 cpmlpmol) in 17 pl of the same buffer as in Figure 1, except that Mg*+ was 12 mM (see Experimental Procedures). Incubation with 10 uM [a-32P]ATP (2700 cpmlpmol) at 38% was continued, and the samples were filtered on membranes.

1 6) PLUS

5. Filter Binding

0.1 dnaA

80

Cell 262

Table 3. Effect the Generation dnaA Protein

of Nucleohde Tightly Bound to dnaA Protein of oriC Sites Sensitive to Pl Nuclease Treatment

Linears

Preincubated with ATP ADP ATPyS Directly incubated with 5 mM ATP No dnaA Preincubated with ATP, omitting subsequent 5 mM ATP

on

(o/o)

45 6 43 41 8

10

dnaA protein (4.8 pmol) was preincubated with 1 uM nucleotide at 0% for 15 min as indicated and added to 150 fmol of supercoiled pCM959 in 60 mM HEPES (pH 6) 320 pglml BSA, 8 mM magnesium acetate, 30% (v/v) glycerol, and 67 ng of HU protein in 50 ul. ATP (5 mM) was included in all but the last line. Following incubation at 38% for 1 min, 1.2. U of Pl nuclease in 30 mM potassium acetate (pH 4.8) was added. PI cleavage was stopped after 5 set by adding 20 ul of 50 mM EDTA and 1% SDS. Densitometric scanning of ethidium bromidestained gels was used to determine the proportion of linear molecules.

sensitivity to the single-strand-specific Pl endonuclease (D. Bramhill and A. Kornberg, unpublished results). The ADP form is specifically blocked at or before this stage (Table 3). In contrast, dnaA protein preincubated with ATP or ATPTS induces a level of Pl sensitivity similar to that of the control directly incubated with 5 mM ATP (see Figure 6 and below). Formation of a Prepriming Complex of oriC with Proteins HU, dna6, and dnaC Is Supported by the ATP Form but Not the ADP Form of dnaA Protein If the block by ADP is at or before the strand separation stage, then the subsequent step should also be blocked. When dnaB and dnaC proteins are present, strand opening by dnaA protein allows entry of dnaB helicase into the DNA at oriC (D. Bramhill and A. Kornberg, unpublished results). This step requires that ATP, 0% template, and

Table 4. ADP Form

of dnaA Protein

Conditions

Incubation

for 38%

Fails to Form

the Prepriming

DNA Synthesis (pmol dNMP)

Exp. 1 No template No dnaA ATP-dnaA ADP-dnaA

O&O 020 210 ? 8 2+2

Exp. 2 No template No dnaA ATP-dnaA ADP-dnaA

1*1 12 + 1 79 + 2 18 % 1

proteins dnaA, dnaB, dnaC, and HU interact at 38%. An active prepriming complex separated from unbound proteins either by gel filtration or density gradient centrifugation shows no further requirement for proteins dnaA, dnaB, dnaC, and HU, and subsequent reactions can occur at 16%. This represents an earlier prepriming event than that reported by van der Ende et al. (1985) and Baker et al. (1986). As measured by replication or by retention of dnaB protein in the oriC complex, the ATP complex of dnaA protein, but not the ADP form, can make the prepriming complex (Table 4). The number of dnaB monomers, estimated per complex, was in the range needed for helicase action by hexamers at the two replication forks in bidirectional replication of the oriC plasmid. ATP in addition to forming a complex with dnaA protein, is required at a concentration of 30 uM for production of the prepriming complex. No other nucleoside triphosphate (i.e., GTP, CTP, UTP dATP, dGTP, dCTP dTTP, ATPyS, and AMPPNP) could be substituted for ATP (data not shown). The dnaB-dnaC protein complex, formation of which is supported by both dATP and ATP (Kobori and Kornberg, 1982) apparently does not explain this absolute ATP requirement. Hydmlysis of ATP Bound to dnaA Protein Is Not Essential for Formation of the Prepriming Complex Formation of the prepriming complex, as measured by subsequent DNA synthesis, reached a maximum within 2 min, corresponding to about one-third of the input template (Figure 6). Complexes formed using [a=P]ATPdnaA protein were isolated by gel filtration. Approximately 7 dnaA protein molecules per input DNA circle (20 per circle replicated) were recovered. Virtually all of the dnaA protein (>90%) remained in the ATP form. The rate of ATP hydrolysis in the presence of the components needed to form the prepriming complex (data not shown) was indistinguishable from that observed without these components added (Figure 2). Clearly, this early stage of ini-

Complex

by the Complex

dnaB

Protein

in the Complex

Measured

Corrected=

35 37 63 27

6 3 9 2

(0) 2 28 0

9&l 12 + 1 36 + 4 13 * 3

(0) 3 27 4

-c -t k -c

dnaA protein was incubated first with 1 uM ATP or 1 HIM ADP. The prepriming reaction with pCM959 DNA, proteins HU, dnaB. and dnaC. and 30 uM ATP was carried out at 38’% for 4 min. In Experiment 1, but not in Experiment 2. omitted components (pCM959 DNA or dnaA protein) were added back after the incubation. The prepriming complex in a 50 ul reaction mixture was separated from free proteins by gel filtration at 4%; the amount of dnaB protein in the void fraction was determined by immunoblotting (Burnette, 1981); the activity of the prepriming complex was measured by a subsequent reaction with SSB, gyrase. primase, and DNA polymerase Ill holoenzyme at 16OC for 30 min. Recovery of the activity by gel filtration was 88 f 3% in Experiment 1 and 57 + 2% in Experiment 2, each with ATP-dnaA. Average values of duplicates normalized for 0.2 ug (0.075 pmol circle) of pCM959 DNA are presented. a Corrected values are listed because measured values include nonspecific binding and/or incomplete separation from free dnaB protein.

ATP Activates 263

E. coli dnaA

Protein

IIIII rATP

2 ATPYS

AMPPNP

0

Figure

6. Kinetics

1

2

3 MINUTES

of the Prepriming

4

5

Reaction

dnaA protein (2.4 pmol) was incubated with 1 uM levels of ATP, ATPTS, AMPPNP, or ADP at O°C for 15 min as in Figure 1, except that Mg2+ was 2.5 mM. The prepriming reaction for the indicated times was carried out at 36% (see Experimental Procedures). As a control, dnaA protein was omitted from the reaction with ATP. DNA synthesis was measured by a subsequent reaction with SSJ, gyrase, primase, and DNA polymerase Ill holoenzyme.

tiation of replication is not accompanied by a significant hydrolysis of the ATP tightly bound to dnaA protein. A complex of the ATP analogue, ATPyS, with dnaA protein was active in the prepriming reaction (Figure 6) and as stable as that with ATF! Over 90% of [sVS]ATP+-dnaA protein complex remained intact after 2 min at 36OC and was very slow to exchange with ATP These results are a further indication that extensive hydrolysis of the tightly bound ATP is not essential for the contribution made by dnaA protein to the prepriming reaction. Discussion Thus far, five ATP-requiring stages in replication of an oriC plasmid have been identified (Baker et al., 1966): transfer of dnaB and dnaC proteins to dnaA protein bound to the oriC sequence, helicase action of dnaB protein, initiation of primers by primase, supercoiling by gyrase, and activa-

Table 5. Homologous Protein

Amino

Acid Sequences

in dnaA

Proteins

Residues

B. subtilis

dnaAb

of E. coli and B. subtilis

and Other

Adenine

Nucleotide-Binding

170-l 65 149-164

Residues identical with the consensus sequences are underlined. a Finch and Emmerson, 1984; consensus of 11 proteins. b Ogasawara et al., 1965.

Proteins

Sequences l/V/L-X-A/G-X-X-X-X-G-K-T-X-X-X-X-X-l/V

ATP bindinga E. coli dnaA

tion of DNA polymerase Ill holoenzyme. Our newest findings extend this list with the ATP dependence of dnaA protein in its action. ATP is bound with extraordinary affinity (Ko = 0.03 PM) to a site on dnaA protein where it protects the protein from inactivation and is hydrolyzed very slowly to ADP. ADP competes with ATP for this site and prevents dnaA protein action in strand separation at oriC (a very early step in initiation) and the subsequent prepriming complex formation with dnaB protein. Thus, the tightly bound ATP is implicated in the performance of an essential replicative function. Several adenine nucleotide-binding proteins share an amino acid consensus sequence (Fable 5). This sequence is found in the dnaA protein of E. coli and in a comparable protein in Bacillus subtilis, despite the fact that these two proteins share less than 50% homology overall (Ogasawara et al., 1965). Whether the B. subtilis protein has a tight binding site for ATP and whether this sequence is the site remain to be determined. Two observations suggest that dnaA protein may have an additional ATP binding site with lower affinity. One is the requirement of a high concentration of ATP (5 mM) for the structural change in the oriC template indicated by sensitivity to the single-strand-specific Pl nuclease (Table 3). The other finding is the need for ATP at a level of at least 30 uM to effect the formation of a prepriming complex of dnaA, dnaB, and dnaC proteins. ATP is known to be required for the formation of a dnaB-dnaC protein complex (Wickner and Hurwitz, 1975), which may be a stage in forming the larger complex; inasmuch as dATP can substitute for ATP in forming the dnaB-dnaC complex (Kobori and Kornberg, 1962) but not the prepriming complex, an additional ATP interaction with dnaA protein may be inferred. Hydrolysis of the tightly bound ATP to ADP is unrelated to the reaction of dnaA protein with dnaB and dnaC proteins in forming the prepriming complex. Formation of the complex is nearly complete after 2 min, when the ATP bound to the dnaA protein is still virtually intact. When the bound ATP is hydrolyzed over the course about 1 hr, the ADP product remains tightly bound to the same site. The ADP form of dnaA protein, whether generated by hydrolysis of ATP or by the addition of ADP to free dnaA protein, is totally inactive in replication. Both the ATP form and the ADP form of dnaA protein bind the four dnaA boxes (9-mers) in oriC and protect them from DNAase I cleavage. Even though these studies were

C-Y-6 I -Y-G

-G-T-G-L-G-K-T-H-L-L-H-A-V_ -G-V-G-L-G-K-T-H-L-M-H-A-l

-

Cell 264

performed with supercoiled DNA, the form essential for the early stage of oriC replication (Funnell et al., 1988), the very nature of the footprinting procedure limits the significance of the results. The DNA is relaxed instantly with the first DNAase cleavage and thus may obscure a difference between complexes of the ATP and ADP forms that requires persistence of the supercoiled state. One striking distinction between the ATP and ADP forms of dnaA protein is the ability of the ATP form to generate sites that are sensitive to cleavage by Pl nuclease. Exposure of phosphodiester bonds in the backbone indicates a conformational change essential for the interaction with the dnaB and dnaC proteins to create the prepriming complex. Also indicative of the distinctive binding of oriC by the ATP form of dnaA protein is that this form, but not the ADP form, generates a complex with oriC and the prepriming assembly that can be visualized with the electron microscope (B. E. Funnell and A. Kornberg, unpublished results). Pertinent in this regard is the binding of dnaA protein to a number of sequences that possess only a single dnaA box (Fuller et al., 1984). Plasmids with such boxes (e.g., F, Rl, and Pl) in their replication origins were previously believed to be independent of drtaA function, but do in fact require dnaA protein for their replication (Hansen and Yarmolinsky, 1986; Ortega and Diaz, 1986). Leakiness of cfnaA mutations may explain the ability of these plasmids to replicate in those mutant strains. For example, in an in vitro system for replication of a miniRl plasmid with one dnaA box in its origin region, only about one-tenth as much dnaA protein is required compared with oriC plasmids (Masai and Arai, 1987); and the ADP, as well as the ATP, form of the protein is active (H. Masai and K. Arai, personal communication). The fate of dnaA protein is still unknown. Whether it is recycled after initiation or is one of the proteins that needs to be synthesized for a new round of replication is an important question. With regard to the inactive ADP form of the protein, it may be so marked for disposal or rejuvenated by exchange with ATP. Were the latter true, the action of factors that regulate the exchange might assume an important role in the initiation of replication. Conceivably, the cellular neighborhood in which dnaA protein is located, perhaps a membrane surface domain containing cardiolipin (observed to destabilize tightly bound nucleotide, K. Sekimizu and A. Kornberg, unpublished results), may influence the function of this pivotal protein. Experimental

Procedures

Reagents Sources were as follows: ATP, deoxyribonucleoside triphosphates, A(p),A (diadenosine tetraphosphate), and HEPES, Pharmacia P-L Biochemicals; GTP, CTP, UTP, AMP, and Tricine. Sigma; adenosine, Calbiochem-Behring; ADP, ATPyS, AMPPNP, and AMPPCP, ICN Pharmaceuticals; [&‘sP]ATP (410 CVmmol) and [@P]dTTP (800 Cilmmol), Amersham Corp.; [Y-~~P]ATP (7000 Cilmmol), New England Nuclear; 1251-labeled protein A (33.4 mCi/mg), ICN Radiochemicals; BSA, Pentex; Bio-Gel A-5m, Bio-Rad. Enzymes Purified replication proteins (Kaguni et al., 1984). dnaA

were prepared as previously described protein was purified by the method de-

scribed previously (Fuller and Kornberg, 1983) with a specific actlvlty of 1.0 x lo6 Ulmg and a purity greater than 90% as judged by SDS-PAGE. Plasmid DNAs pCM959 (Meijer et al., 1979), a gift from M. Meijer (University of Amsterdam, The Netherlands), is a minichromosome (4012 bp) consisting solely of E. coli DNA encompassing oriC (bp -677 to +3335). M13oriC26 and Ml3oriC2LB5 contain oricsequences in M13AE101, an Ml3 derivative lacking the complementary strand origin (Kaguni and Kornberg, 1984). M13mpRE85 (Smith et al., 1985) (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 Pstl site of Ml3mp8. Supercoiled plasmid DNAs were prepared as previously described (Ogawa et al., 1985). ATP(ADP)-Binding Assay [a-32P]ATP (5 x 10s to 1 x 104 cpmlpmol) or [‘4C]ADP (750 cpmlpmol) bound to dnaA protein was determined by adsorption to nitrocellulose membrane filters (Millipore HA 0.45 PM. 24 mm diam.). The standard reaction (40 ~1) contained 50 mM Tricine-KOH (pH 8.25 at 1 M), 0.5 mM magnesium acetate, 0.3 mM EDTA, 20% (v/v) glycerol, 0.007% Triton X-100, 7 mM DTT, 1 PM [a-=P]ATP or [‘4c]ADc and 120-240 ng of dnaA protein. After incubation at O°C for 15 min, the solution was filtered through a nitrocellulose membrane filter presoaked in wash buffer (50 mM Tricine-KOH [pH 8.25 at 1 M], 0.5 mM magnesium acetate, 0.3 mM EDTA, 5 mM DTT, 10 mM (NH&S04, 17% [vhr] glycerol, 0.005% Triton X-100). The filter was washed with 6 ml of ice-cold wash buffer and dried under an infrared lamp; radioactivity retained on the filter was measured in a liquid scintillation counter. The reaction without dnaA protein provided a background value of 0.05 pmol. The Ko of ATP-dnaA protein and the number of ATP binding sites were calculated according to Scatchard (1949). Reconstitution Assay for DNA Replication The standard reaction (25 ~1) contained 40 mM HEPES-KOH (pH 7.6), 8 mM magnesium acetate, 13% (v/v) glycerol, 0.004% Triton X-100, 2 mM phosphocreatine, 2 mM DTT, 2 mM ATP, 500 pM GTP, CTI? and UTP, each, 100 WM dATP, dGTP, and dCTP, each, 100 NM (30-150 cpmlpmol) [@P]dTTP, 200 ng (600 pmol of nucleotide) pCM959 DNA, 500 ng of creatine kinase, 120 ng of dnaA protein, 60 ng of dnaB protein, 20 ng of dnaC protein, 20 ng of HU, 640 ng of SSB, 300 ng of gyrase A subunit, 150 ng of gyrase B subunit, 10 ng of primase, 70 ng of DNA polymerase Ill (pol Ill) holoenzyme, and 75 ng of purified b subunit to complement a deficiency in the pol Ill fraction. When RNA polymerase (2900 ng) was added, glycerol and Triton X-100 were replaced with 0.4 mglml BSA, a higher level of HU protein (200 ng), and RNAase H (1.4 ng) (Ogawa et al., 1985). The mixtures were assembled at O°C and incubated at 30°C. Total nucleotide incorporation (pmol) was measured in a liquid scintillation counter after trichloroacetic acid precipitation onto Whatman GWC glass-fiber filters. To compare the activities of the ATP form and the ADP form of dnaA protein, the protein (120 ng) was first incubated with 1 FM ATP or ADP in the absence of template DNA and other replication proteins at O°C for 15 min. The omitted components were then added, and the reaction was continued. To form the prepriming complex, the mixture (19 ~1) containing 2.5 mM magnesium acetate, 30 KM ATP, template DNA, and proteins HU, dnaA, dnaB, and dnaC, was incubated at 38OC. The prepriming reaction was terminated by chilling the sample on ice. For measuring replication activity, the proteins (gyrase, SSB. primase, and pol Ill), magnesium acetate (to 8 mM), ATP (to 2 mM), GTP, CTP, UTP, dATP, dGTP, dCTP, and [a-32P]dTTP were added and DNA synthesis was carried out at 16OC for 30 min. Measurement of the Uptake of dnaB Protein into the oriC Complex The prepriming complex was separated from free proteins by spin-gel filtration through columns of Bio-Gel A-5m in 40 mM Tricine-KOH (pH 8.25 at 1 M), 2.5 mM magnesium acetate, 0.1 mM EDTA, 17% (v/v) glycerol, 5 mM OTT, 0.15 mM ATP, 0.005% Briton X-100, and 10 mM (NH&SO4 packed in a 1 ml plastic syringe essentially as described (Maniatis et al., 1982). A sample of the DNA-containing fractions was subjected to SDS-PAGE on 12.5% gels). Proteins were blotted to a

ATP Activates

E. coli dnaA

Protein

265

nitrocellulose filter in 20 mM Tris, 156 mM glycine, 29% (v/v) methanol by using the Bio-Rad blotting apparatus. The filter was incubated with antidnaB protein antibody and 1251-labeled protein A, washed, and then subjected to autoradiography (Burnette. 1981). The amount of dnaB protein was calculated from densitometric scanning of the film and appropriate standards.

Acknowledgments We thank H. Masai and K. Arai for communication of results prior to publication. We are grateful to L. Bertsch for careful reading of the manuscript. This research was supported by grants from the National Institutes of Health and the National Science Foundation. K. S. is a Fellow of the American Cancer Society. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “a’advertisement” in accordance with 16 USC. Section 1734 solely to indicate this fact. Received

April 17, 1967.

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