Jon M. Kaguni

Ordered and sequential binding of DnaA protein to oriC, the chromosomal origin of Escherichia coli

DnaA protein of Escherichia coli acts in initiation of chromosomal DNA replication by binding specific sequences, termed DnaA boxes in the chromosomal origin, oriC. On binding, it induces a localized unwinding to create a structure recognized by other replication proteins that act subsequently in the initiation process. In this report, we examined the binding of DnaA protein to each of the DnaA boxes in oriC. By gel mobility shift assays, DnaA protein formed at least six discrete complexes. ATP or ADP included in the reaction mixture prior to electrophoresis was required. Chemical cleavage of isolated complexes with 1,10-phenanthroline-copper revealed that DnaA protein binds in an ordered manner to the DnaA boxes in oriC. Preferential binding to one DnaA box (R4) was confirmed by demonstration that a DNA fragment containing it was bound with greater affinity than another DnaA box sequence (R1). In vitro replication activity correlated with a complex formed at a ratio of 30 DnaA monomers/oriC in which all DnaA boxes are occupied. The last site bound is DnaA box R3. This event may be critical in promoting initiation from oriC as it correlates with in vivo observations that binding of DnaA protein to box R3 occurs at the time of initiation of chromosomal replication, whereas other DnaA boxes are bound by DnaA protein throughout the cell cycle (Cassler, M. R., Grimwade, J. E., and Leonard, A. C. (1995) EMBO J. 14, 5833-5841).

Hyperinitiation of DNA replication in Escherichia coli leads to replication fork collapse and inviability

Elevated dnaA expression from a multicopy plasmid induces more frequent initiation from the Escherichia coli replication origin, oriC, but viability is maintained. In comparison, chromosomally encoded dnaAcos also stimulates initiation, but this is lethal. By quantitative methods, we show that the level of initiation induced by elevated dnaA expression leads to collapsed replication forks that are mostly within 10 map units of oriC. Because forks collapse randomly, nucleoprotein complexes at specific sites such as datA are not the cause. When replication restart is blocked by a mutation in recB or priA, the increased initiations via elevated dnaA expression causes inviability. The amount of collapsed forks is substantially higher under elevated expression of dnaAcos compared to that of dnaA. We propose that the lethal phenotype of chromosomally encoded dnaAcos is a result of hyperinitiation that overwhelms the repair capacity of the cell.

Helicase Loading at Chromosomal Origins of Replication

Loading of the replicative DNA helicase at origins of replication is of central importance in DNA replication. As the first of the replication fork proteins assemble at chromosomal origins of replication, the loaded helicase is required for the recruitment of the rest of the replication machinery. In this work, we review the current knowledge of helicase loading at Escherichia coli and eukaryotic origins of replication. In each case, this process requires both an origin recognition protein as well as one or more additional proteins. Comparison of these events shows intriguing similarities that suggest a similar underlying mechanism, as well as critical differences that likely reflect the distinct processes that regulate helicase loading in bacterial and eukaryotic cells.

DnaA Protein of Escherichia coli: Oligomerization at the E. coli chromosomal origin is required for initiation and involves specific N-terminal amino acids

Iterated DnaA box sequences within the replication origins of bacteria and prokaryotic plasmids are recognized by the replication initiator, DnaA protein. At the E. coli chromosomal origin, oriC, DnaA is speculated to oligomerize to initiate DNA replication. We developed an assay of oligomer formation at oriC that relies on complementation between two dnaA alleles that are inactive by themselves. One allele is dnaA46; its inactivity at the non‐permissive temperature is due to a specific defect in ATP binding. The second allele, T435K, does not support DNA replication because of its inability to bind to DnaA box sequences within oriC. We show that the T435K allele can complement the dnaA46(Ts) allele. The results support a model of oligomer formation in which DnaA box sequences of oriC are bound by DnaA46 to which T435K then binds to form an active complex. Relying on this assay, leucine 5, tryptophan 6 and cysteine 9 in a predicted alpha helix were identified that, when altered, interfere with oligomer formation. Glutamine 8 is additionally needed for oligomer formation on an oriC‐containing plasmid, suggesting that the structure of the DnaA‐oriC complex at the chromosomal oriC locus is similar but not identical to that assembled on a plasmid. Other evidence suggests that proline 28 of DnaA is involved in the recruitment of DnaB to oriC. These results provide direct evidence that DnaA oligomerization at oriC is required for initiation to occur.

Replication initiation at the Escherichia coli chromosomal origin

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.

The box VII motif of Escherichia coli DnaA protein is required for DnaA oligomerization at the E. coli replication origin.

Escherichia coli DnaA protein initiates DNA replication from the chromosomal origin, oriC, and regulates the frequency of this process. Structure-function studies indicate that the replication initiator comprises four domains. Based on the structural similarity of Aquifex aeolicus DnaA to other AAA+ proteins that are oligomeric, it was proposed that Domain III functions in oligomerization at oriC (Erzberger, J. P., Pirruccello, M. M., and Berger, J. M. (2002) EMBO J. 21, 4763–4773). Because the Box VII motif within Domain III is conserved among DnaA homologues and may function in oligomerization, we substituted conserved Box VII amino acids of E. coli DnaA with alanine by site-directed mutagenesis to examine the role of this motif. All mutant proteins are inactive in initiation from oriC in vivo and in vitro, but they support RK2 plasmid DNA replication in vivo. Thus, RK2 requires only a subset of DnaA functions for plasmid DNA replication. Biochemical studies on a mutant DnaA carrying an alanine substitution at arginine 281 (R281A) in Box VII show that it is inactive in in vitro replication of an oriC plasmid, but this defect is not from the failure to bind to ATP, DnaB in the DnaB-DnaC complex, or oriC. Because the mutant DnaA is also active in the strand opening of oriC, whereas DnaB fails to bind to this unwound region, the open structure is insufficient by itself to load DnaB helicase. Our results show that the mutant fails to form a stable oligomeric DnaA-oriC complex, which is required for the loading of DnaB.

Domains of DnaA Protein Involved in Interaction with DnaB Protein, and in Unwinding the Escherichia coli Chromosomal Origin

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 protein-binding 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.

Escherichia coli DnaA interacts with HU in initiation at the E. coli replication origin.

Escherichia coli HU protein is a dimer encoded by two closely related genes whose expression is growth phase‐dependent. As a major component of the bacterial nucleoid, HU binds to DNA non‐specifically, but acts at the chromosomal origin (oriC) during initiation by stimulating strand opening in vitro. We show that the α dimer of HU is more active than other forms of HU in initiation of an oriC‐containing plasmid because it more effectively promotes strand opening of oriC. Other results demonstrate that HU stabilizes the DnaA oligomer bound to oriC, and that the α subunit of HU interacts with the N‐terminal region of DnaA. These observations support a model whereby DnaA interacts with the α dimer or the αβ heterodimer, depending on their cellular abundance, to recruit the respective form of HU to oriC. The greater activity of the α dimer of HU at oriC may stimulate initiation during early log phase compared with the lesser activity of the αβ heterodimer or the β dimer.

Transcriptional repression of the dnaA gene of Escherichia coli by dnaA protein

SummaryThe promoter region of the dnaA gene and of a gene which encodes a 16 kDa protein contain sites which are recognized and bound by dnaA protein. Using assays of run-off transcription of restriction fragments, purified dnaA protein specifically repressed transcription from both dnaA promoters and from the promoter for the 16 KD gene to almost undetectable levels. This repressive effect was observed at levels of dnaA protein required for specific binding of dnaA protein to restriction fragments containing the promoters for these genes. These results indicate that transcription of these genes is regulated by binding of dnaA protein to the promoter regions of these genes.

Threonine 435 of Escherichia coli DnaA protein confers sequence-specific DNA binding activity.

The Escherichia coli DnaA protein, as a sequence-specific DNA binding protein, promotes the initiation of chromosomal replication by binding to four asymmetric 9-mer sequences termed DnaA boxes in oriC. Characterization of N-terminal, C-terminal, and internal in-frame deletion mutants identified residues near the C terminus of DnaA protein required for DNA binding. Furthermore, genetic and biochemical characterization of 11 missense mutations mapping within the C-terminal 89 residues indicated that they were defective in DNA binding. Detailed biochemical characterization of one mutant protein bearing a threonine to methionine substitution at position 435 (T435M) revealed that it retained only nonspecific DNA binding activity, suggesting that threonine 435 imparts specificity in binding. Finally, T435M was inactive on its own for in vitro replication of an oriC plasmid but was able to augment limiting levels of wild type DnaA protein, consistent with the proposal that not all of the DnaA monomers in the initial complex are bound specifically to oriC and that direct interaction occurs among monomers.