DnaC, the indispensable companion of DnaB helicase, controls the accessibility of DnaB helicase by primase.

Former studies relying on hydrogen/deuterium exchange analysis suggest that DnaC bound to DnaB alters the conformation of the N-terminal domain (NTD) of DnaB to impair the ability of this DNA helicase to interact with primase. Supporting this idea, the work described herein based on biosensor experiments and enzyme-linked immunosorbent assays shows that the DnaB-DnaC complex binds poorly to primase in comparison with DnaB alone. Using a structural model of DnaB complexed with the C-terminal domain of primase, we found that Ile-85 is located at the interface in the NTD of DnaB that contacts primase. An alanine substitution for Ile-85 specifically interfered with this interaction and impeded DnaB function in DNA replication, but not its activity as a DNA helicase or its ability to bind to ssDNA. By comparison, substitutions of Asn for Ile-136 (I136N) and Thr for Ile-142 (I142T) in a subdomain previously named the helical hairpin in the NTD of DnaB altered the conformation of the helical hairpin and/or compromised its pairwise arrangement with the companion subdomain in each brace of protomers of the DnaB hexamer. In contrast with the I85A mutant, the latter were defective in DNA replication due to impaired binding to both ssDNA and primase. In view of these findings, we propose that DnaC controls the ability of DnaB to interact with primase by modifying the conformation of the NTD of DnaB.

Mutant DnaAs of Escherichia coli that are refractory to negative control.

DnaA is the initiator of DNA replication in bacteria. A mutant DnaA named DnaAcos is unusual because it is refractory to negative regulation. We developed a genetic method to isolate other mutant DnaAs that circumvent regulation to extend our understanding of mechanisms that control replication initiation. Like DnaAcos, one mutant bearing a tyrosine substitution for histidine 202 (H202Y) withstands the regulation exerted by datA, hda and dnaN (ß clamp), and both DnaAcos and H202Y resist inhibition by the Hda-ß clamp complex in vitro. Other mutant DnaAs carrying G79D, E244K, V303M or E445K substitutions are either only partially sensitive or refractory to inhibition by the Hda-ß clamp complex in vitro but are responsive to hda expression in vivo. All mutant DnaAs remain able to interact directly with Hda. Of interest, both DnaAcos and DnaAE244K bind more avidly to Hda. These mutants, by sequestrating Hda, may limit its availability to regulate other DnaA molecules, which remain active to induce extra rounds of DNA replication. Other evidence suggests that a mutant bearing a V292M substitution hyperinitiates by escaping the effect of an unknown regulatory factor. Together, our results provide new insight into the mechanisms that regulate replication initiation in Escherichia coli.

Two forms of ribosomal protein L2 of Escherichia coli that inhibit DnaA in DNA replication.

We purified an inhibitor of oriC plasmid replication and determined that it is a truncated form of ribosomal protein L2 evidently lacking 59 amino acid residues from the C-terminal region encoded by rplB. We show that this truncated form of L2 or mature L2 physically interacts with the N-terminal region of DnaA to inhibit initiation from oriC by apparently interfering with DnaA oligomer formation, and the subsequent assembly of the prepriming complex on an oriC plasmid. Both forms of L2 also inhibit the unwinding of oriC by DnaA. These in vitro results raise the possibility that one or both forms of L2 modulate DnaA function in vivo to regulate the frequency of initiation.

An essential tryptophan of Escherichia coli DnaA protein functions in oligomerization at the E. coli replication origin.

In the initiation of bacterial DNA replication, DnaA protein recruits DnaB helicase to the chromosomal origin, oriC, leading to the assemble of the replication fork machinery at this site. Because a region near the N terminus of DnaA is required for self-oligomerization and the loading of DnaB helicase at oriC, we asked if these functions are separable or interdependent by substituting many conserved amino acids in this region with alanine to identify essential residues. We show that alanine substitutions of leucine 3, phenylalanine 46, and leucine 62 do not affect DnaA function in initiation. In contrast, we find on characterization of a mutant DnaA that tryptophan 6 is essential for DnaA function because its substitution by alanine abrogates self-oligomerization, resulting in the failure to load DnaB at oriC. These results indicate that DnaA bound to oriC forms a specific oligomeric structure, which is required to load DnaB helicase.

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.