An enzyme system that replicates plasmids bearing the origin of the Escherichia coli chromosome (oriC) has the following physiologically relevant features. The system (i) depends completely on low levels ofexogenously furnished supercoiled oriC plasmids, (ii) uses only those plasmids that contain the intact oriC region of about 245 base pairs, (iii) initiates replication within or near the oniC sequence and proceeds bidirectionally, (iv) proceeds linearly, after a 5-min lag, for 30-40 min to produce as much as a 40% increase over the input DNA, (v) depends on RNA polymerase and gyrase as indicated by total inhibition by rifampicin and nalidixate, (vi) depends on replication proteins (e.g., dnaB protein and single-stranded DNA binding protein) as judged by specific antibody inhibitions, (vii) operates independently from protein synthesis, and (viii) depends on dnaA activity, as suggested by the inactivity ofenzyme fractions from each oftwo dnaA temperaturesensitive mutant strains, and complementation (with a 15-fold overproduction of complementing activity) by a fraction from a strain containing the dnaA gene cloned in a multicopy plasmid. Resolution and analysis of factors that control the initiation of a chromosome cycle should become accessible through this enzyme system. Initiation of a new cycle ofchromosomal replication is the most critical step in the control of replication, and an enormous amount ofeffort has been expended to understand it (1, 2). Yet little is known about the mechanism of the initiation of chromosomal replication because biochemical data are extremely scant.In Escherichia coli, several genes, including dnaA, dnaI, and dnaP, have been identified as essential for initiating a cycle of replication (1, 2). The denatured dnaA polypeptide has been detected (e.g., ref. 3), but nothing is known about the products of dnal and dnaP. Genes dnaB and dnaC, whose products are essential for ongoing replication, are also needed at or near the outset ofa cycle ofchromosome replication (1, 2). The inhibitory effects of rifampicin at this stage of replication have also implicated RNA polymerase (1, 2).Once initiated, propagation of the replication fork is relatively well understood through the actions of the multisubunit DNA polymerase III holoenzyme (4) and a primosomal unit composed ofperhaps seven discrete proteins (5). Isolation ofthis cellular machinery came through the use of small, singlestranded DNA phage chromosomes as probes to illuminate the enzymatic components and their detailed operations. Enzyme systems that replicate plasmids have also made it possible to demonstrate direct priming by RNA polymerase and copy-number control by small RNAs for ColEl (6), and a requirement for positive, trans-acting, plasmid-encoded proteins for R6K (7) and R1 (8).The availability of the chromosomal origin of E. coli (oriC) in a functional form as part of a small plasmid (9) and earlier success in the enzymatic resolution of phage replication have sustained our efforts for several years to discover a...

Escherichia coli is a model system to study the mechanism of DNA replication and its regulation during the cell cycle. One regulatory pathway ensures that initiation of DNA replication from the chromosomal origin, oriC, is synchronous and occurs at the proper time in the bacterial cell cycle. A major player in this pathway is SeqA protein and involves its ability to bind preferentially to oriC when it is hemi-methylated. The second pathway modulates DnaA activity by stimulating the hydrolysis of ATP bound to DnaA protein. The regulatory inactivation of DnaA function involves an interaction with Hda protein and the beta dimer, which functions as a sliding clamp for the replicase, DNA polymerase III holoenzyme. The datA locus represents a third mechanism, which appears to influence the availability of DnaA protein in supporting rifampicin-resistant initiations.

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To initiate DNA replication, DnaA recognizes and binds to specific sequences within the Escherichia coli chromosomal origin (oriC), and then unwinds a region within oriC. Next, DnaA interacts with DnaB helicase in loading the DnaB-DnaC complex on each separated strand. Primer formation by primase (DnaG) induces the dissociation of DnaC from DnaB, which involves the hydrolysis of ATP bound to DnaC. Recent evidence indicates that DnaC acts as a checkpoint in the transition from initiation to the elongation stage of DNA replication. Freed from DnaC, DnaB helicase unwinds the parental duplex DNA while interacting the cellular replicase, DNA polymerase III holoenzyme, and primase as it intermittently forms primers that are extended by the replicase in duplicating the chromosome.

DnaA protein of Escherichia coli is a sequence-specific DNA-binding protein required for the initiation of DNA replication from the chromosomal origin, oriC. It is also required for replication of several plasmids including pSC101, F, P-1, and R6K. A collection of monoclonal antibodies to DnaA protein has been produced and the primary epitopes recognized by them have been determined. These antibodies have also been examined for the ability to inhibit activities of DNA binding, ATP binding, unwinding of oriC, and replication of both an oriC plasmid, and an M13 single-stranded DNA with a proposed hairpin structure containing a DnaA proteinbinding site. Replication of the latter DNA is dependent on DnaA protein by a mechanism termed ABC priming. These studies suggest regions of DnaA protein involved in interaction with DnaB protein, and in unwinding of oriC, or low-affinity binding of ATP.DnaA protein of Escherichia coli is required for the initiation of chromosomal replication from oriC, the chromosomal origin (1-3). As a DNA-binding protein, it recognizes a 9-base pair sequence, the DnaA box, present 4 times in oriC (3). On binding, localized melting of the AT-rich region by DnaA protein and 1-5 mM ATP (4) is assisted by either HU or IHF that presumably act by DNA bending (5-7). Subsequently, the binding of DnaB helicase from the DnaB-DnaC complex through direct contact with DnaA protein suggests that it orients the binding of the replicative helicase for its subsequent action to promote bidirectional replication fork movement (8). Priming of DNA replication by primase for both leading and lagging strand synthesis is proposed to occur through a physical interaction between primase and DnaB protein (9), thus establishing the elongation phase of DNA replication.The correlation of domains of DnaA protein to its various functions is rudimentary. By comparative analysis of homologs of the dnaA gene from phylogenetically diverse microbes, sequence conservation of a P-loop motif, GX 4 GKT located at residues 172-179 (reviewed in Ref. 10), correlates with the activity of DnaA protein to bind ATP with high affinity (K D ϳ 0.03 M) (11). In p21 ras and RecA protein, corresponding residues interact with the phosphates of the bound nucleotide (12, 13). Missense mutations of the dnaA5 and dnaA46 alleles, as well as others (14), substitute a conserved alanine for valine at amino acid residue 184 near the P-loop. The mutant proteins were defective in ATP binding, suggesting that the substitution was responsible (15, 16).1 Together, these results implicate this region of DnaA protein in binding to ATP.The DNA-binding domain of DnaA protein has been localized to a region from residue 379 to the carboxyl terminus (17). Other domains of DnaA protein involved in purine binding, unwinding, interaction with DnaB protein (8), or interaction with phospholipids that displace the bound nucleotide (18) are unknown. To correlate domains of DnaA protein to its various functions, a collection of monoclonal antibodies were produced and characte...