Fate of the DnaA initiator protein in replication at the origin of the Escherichia coli chromosome in vitro.

The dnaA initiator protein binds specific sequences in the 245-base pair Escherichia coli origin (oriC) to form a series of complexes which eventually are opened enough to admit dnaB helicase into a prepriming complex (Bramhill, D., and Kornberg, A. (1988) Cell 52, 743-755). ATP bound to a high-affinity site on dnaA protein is the preferred form for one or more of the early stages, but an elevated level of ATP is needed for a later stage; further evidence for a low-affinity site has now been obtained. We find that at limiting levels of dnaA protein only the ATP form produces an active initial complex; neither the ADP nor the non-nucleotide forms are effective. Augmentation of the activity of a limiting level of the ATP form of dnaA protein by the otherwise inert ADP form implies that at some stage of initiation both forms are active. The dnaA protein is essential up to the stage of forming the prepriming complex; upon salt dissociation from an oriC complex, the protein can be recycled to function at a fresh origin. Distinctive conformational states of the ATP form are implied by interactions with oriC DNA, by the influence of phospholipids on accelerating nucleotide exchange, and by the susceptibility to proteolytic cleavage.

The dnaA initiator protein binds specific sequences in the 245-base pair Escherichia coli origin (oriC) to form a series of complexes which eventually are opened enough to admit dnaB helicase into a prepriming complex  Cell 52, 743-755).
ATP bound to a high-affinity site on dnaA protein is the preferred form for one or more of the early stages, but an elevated level of ATP is needed for a later stage; further evidence for a low-affinity site has now been obtained.
We find that at limiting levels of dnaA protein only the ATP form produces an active initial complex; neither the ADP nor the non-nucleotide forms are effective. 1n10 a m~crocentr~lu~e tut)e. Repeatedly. an equal volume of' column buffer ('LOO pII \vaS applied and the gel aa$ centrif'uged again lor each suCCehhl\ e I ract 1on. dissociated dnaA protein from the prepriming complex (at the next stage) (4, 1.5) as effectively as from the open complex, the loss of dnaA protein had little effect on the capacity of the oriC DNA to support replication (Fig. S); further addition of dnaA, HU, dnaB, and dnaC proteins was not required. Thus, dnaA protein is dispensable beyond the prepriming complex for all the subsequent stages of initiation, priming, and synthesis.

RESULTS
Only the A7'P Form of DnaA Protein Produces nn Active Initial Complex u:ith oriC-Although dnaA protein in any form (i.e. ATP, ADP, or non-nucleotide) produces a complex with oriC DNA at 0 "C, t,he isolation of a stable, replicationactive complex required that the ATP form be maintained at an elevated temperature (e.g. 37 "C) (15). By replacing the sucrose gradient technique, which consumes several hours, with gel filtration, which takes only a few minutes, an active replication complex could be separated even at 0 "C. The yield of active complex was near 80% with the ATP form (Fig. l), but less than 10% with either the ADP or non-nucleotide forms (data not shown).
DnaA Protein Is Required for Formation of the Open and Prepriming Complexes Rut Not ReJpond-The open complex of dnaA protein with oriC (4, 15), formed with ""S-labeled protein in the presence of HU protein and 5 mM ATP at :38 "C, was isolated by rapid gel filtration.
Virtually none of the complex remained after exposure to salt (e.g. 200 mM K+glutamate) (Fig. 2). Free ""S-dnaA protein was not detect.ed in the elution profile. Possibly, due to the low level of protein employed, the dissociated dnaA protein was nonspecifically absorbed by the gel matrix. Whereas salt treatment also DnaA Protein Can He Recycled in oriC Replication-The ability of dnaA protein to function again after prepriming complex formatlon with pCM959 was demonstrated by challenge with another oriC template (i.e. RE85). Because these plasmids differ in size, their replication products were readily separated by electrophoresis.
Prepriming complexes formed with pCM959 were isolated by gel filtration.
Upon addition of replication enzymes (but no dnaA protein) and the challenge template (RE85) to the prepriming complex, the replication products analyzed by agarose gel electrophoresis included both the challenge and initial templates (Fig. 4) Influence of ATP on Tryptic Proteolysis of DnaA Protein in the Early Complexes-Digestion with trypsin at 37 "C of ATP-dnaA protein complexed with oriC DNA resulted in a prompt loss of replication activity (Fig. 5) along with the disappearance of the 55-kDa band (11) of the protein on SDS-polyacrylamide gel electrophoresis (data not shown). However, the oriC DNA bound to the protein was largely retained. Under these digestion conditions, over 80% of the ATP-dnaA protein in the complex was converted to an NH2-terminal 30-kDa fragment which resisted further digestion for over 2 h (data not shown). By comparison, the 30-kDa fragment from an ADP . dnaA protein complex was digested to smaller fragments within 1 h (data not shown). A strikingly different digestion pattern was observed with the non-nucleotide dnaA protein complex; the 30-kDa as well as the 55-kDa peptide band disappeared within 5 min.
Even with much milder proteolysis (IOO-fold less trypsin), the nucleotide forms of dnaA protein complexed with oriC DNA were readily converted to the 30-kDa fragment, whereas the nucleotide-free protein (not complexed with oriC DNA) remained undigested (data not shown). We may infer that conformational changes attendant upon binding of ATP-dnaA or ADP-dnaA protein to oriC DNA expose the COOHterminal region of the protein to tryptic cleavage.

Elevated Levels of the ADP Form of DnaA Protein Exhibit Replication
Activity-Based on filter retention experiments (2), 20-40 dnaA protein molecules bind per origin sequence; a similar ratio is required for optimal Pl nuclease linearization of an oriC-containing plasmid (4). The dnaA protein-oriC complex observed in the electron microscope (3,16) also suggests 20-40 dnaA protein monomers enveloped by DNA. At levels of dnaA protein approaching this stoichiometry, only the ATP form of the protein had replication activity, while the ADP form was inert (Fig. 6). However, at a 3-fold greater level, the ADP form was active for replication indicating a clear preference for the ATP form for one or more of the early stages in the initiation of replication. Low Levels of the ADP Form of DnaA Protein Augment the Replication Activity of the ATP Form-When the ADP form was mixed with the ATP form, it neither competed nor inhibited but augmented the activity of a limiting level of the ATP form (Fig. 7). Similar augmentation was observed when the nucleotide forms of dnaA protein were formed in the presence of 100 PM ATP and ADP (data not shown). No replication activity was observed when comparable quantities of ADP-dnaA protein were mixed with ATP (1 or 100 PM)   I  I  I  I  I  I  I  lo  0  1  2  3  4  5  6   (3 pmol) at 0 "C, either 10 min before the addition or, as a control, along with the addition of pBSoriC and other replication components.
The replication reaction for the indicated times was at 30 "C (see "Materials and Methods").
alone (data not shown). Thus, among the several stages at which dnaA protein functions, the ATP form is more efficient for some, while both forms are active at others. The Low Affinity Site for Nucleotides on DnnA Protein-Beyond the very tight binding of a nucleotide (ATP or ADP) with a I& of 0.1 PM or less (5), dnaA protein requires high levels of ATP (e.g. near 5 mM) to form the open oriC complex (4). Further insight into nucleotide binding at this implied low affinity site is provided by the action of high levels of ADP. When the ATP form of dnaA protein was exposed to 4 mM ADP for 10 min at 0 "C, the protein was severely inhibited in responding to the high level of ATP to form the open complex assayed by replication (Fig. 8). Presumably, this ADP occupancy of the low affinity ATP site is responsible for the inactivation. DISCUSSION In the course of opening the duplex of the E. coli chromosome at its unique origin, oriC, the dnaA protein undergoes a number of conformational changes including aggregation into a sphere of 20 or so molecules (3). Binding of ATP to a high affinity site on the protein (5), we now find, is the most effective form for achieving the earliest active stage that succeeds the initial binding of dnaA protein to its four 9-mer recognition sequences in oriC (Fig. 1). Despite the apparent similarity of the oriC DNA footprints of the ATP, ADP, and non-nucleotide forms of dnaA protein3 the capacity to form an active initial complex requires ATP.
To proceed to the open and then to the prepriming complexes, dnaA protein requires HU protein, a high level of ATP (e.g. 5 mM) and an elevated temperature (>25 "C) (4,11). The stages beyond the prepriming complex proceed effectively despite the removal of virtually all the dnaA protein (Figs. 2 and 3). Upon dissociation of dnaA protein from the prepriming complex, effected by salt, the protein can be recycled to a fresh oriC template (Fig. 4). Whether recycling occurs in uiuo is uncertain.
With ATP tightly bound, dnaA protein interacts with and opens a sequence of three 13-mers adjoining the 9-mer region in oriC (4,11). Conformational changes in dnaA protein, not achieved with ADP binding, are implied by altered susceptibility to proteolytic cleavage. Binding of the ATP form of dnaA protein exposes its COOH-terminal region to tryptic action, conceivably a structural change related to the entry of the dnaB helicase (from the dnaB . dnaC complex) into the prepriming complex. The ADP form of dnaA protein, though ineffective in forming the initial complex (data not shown) and supporting oriC replication (Fig. 6), augments rather than inhibits the activity of limiting levels of the ATP form (Fig. 7). From this result, we infer that while the ATP form is required for one or more of the several stages of initiation, the ADP form may 3 K. Sekimizu, D. Bramhill, and A. Kornberg, unpublished data.
be substituted at some stage, and at high levels, is effective at all stages (Fig. 6). Consistent with this interpretation is the ability of the ADP form to support the replication of Rl plasmids, the origins of which possess a single dnaA box. 4 The numerous molecules of dnaA protein involved in initiation and their several functions dependent on ATP imply complex binding kinetics of ATP to the protein.
Beyond occupancy of the high affinity site (I& = 0.03 PM) for producing the initial complex, the requirement for a high level of ATP (near 5 mM) for proceeding to the open and prepriming complex stages implies a low-affinity site. Direct evidence for this site has been difficult to obtain, but the striking inactivation by a high level of ADP of the protein with its highaffinity site occupied by ATP, is a clear indication of an irreversible occupancy of such a low affinity site (Fig. 8).