DiaA, a Novel DnaA-binding Protein, Ensures the Timely Initiation of Escherichia coli Chromosome Replication

Abstract

The DnaA protein is the initiator of Escherichia coli chromosomal replication. In this study, we identify a novel DnaA-associating protein, DiaA, that is required for the timely initiation of replication during the cell cycle. DiaA promotes the growth of specific temperature-sensitive dnaA mutants and ensures stable minichromosome maintenance, whereas DiaA does not decrease the cellular DnaA content. A diaA::Tn5 mutation suppresses the cold-sensitive growth of an overinitiation type dnaA mutant independently of SeqA, a negative modulator of initiation. Flow cytometry analyses revealed that the timing of replication initiation is disrupted in the diaA mutant cells as well as wild-type cells with pBR322 expressing the diaA gene. Gel filtration and chemical cross-linking experiments showed that purified DiaA forms a stable homodimer. Immunoblotting analysis indicated that a single cell contains about 280 DiaA dimers. DiaA stimulates minichromosome replication in an in vitro system especially when the level of DnaA included is limited. Moreover, specific and direct binding between DnaA and DiaA was observed, which requires a DnaA N-terminal region. DiaA binds to both ATP-and ADP-bound forms of DnaA with a similar affinity. Thus, we conclude that DiaA is a novel DnaAassociating factor that is crucial to ensure the timely initiation of chromosomal replication.

Importance of Proteins Controlling Initiation of DNA Replication in the Growth of the High-Pressure-Loving Bacterium Photobacterium profundum SS9

“…Consequently, E. coli DiaA acts as a positive regulator of the initiation of DNA replication. In the absence of DiaA, initiation of DNA replication is delayed and in E. coli cells with two oriC copies, it only occurs from one of these, resulting in cells with three copies of their chromosome (14). In contrast, this is an extremely rare occurrence in wild-type E. coli cells.…”

“…In E. coli, DiaA is necessary to ensure the timely initiation of DNA replication (14). DiaA forms a tetramer and binds to multiple molecules of DnaA, promoting (i) the binding of DnaA to the origin of replication in E. coli (known as oriC), (ii) ATP-DnaA-specific conformational changes in the oriC complex, and (iii) the unwinding of oriC DNA (17).…”

“…One of the putative high-pressure-sensitive mutants (FL23) isolated from these screens had a mini-Tn10 insertion in the gene PBPRA3229, which encodes a protein with 75% identity (85% similarity) to Escherichia coli DiaA (DnaA initiator-associating factor) (14). Although FL23 shows growth defects at 0.1 MPa (15°C) relative to the parent strain, the ratio of growth at 45 MPa to growth at 0.1 MPa and 15°C is substantially reduced compared to that of the parent strain, confirming that disruption of PBPRA3229 results in a high-pressure sensitivity growth phenotype (19).…”

NMR structure of the N-terminal domain of the replication initiator protein DnaA

“…Other experiments have suggested a role for the ATPase domain in oligomerization [6,7,15, 16,20]. Together, the observations that full-length DnaA from E.coli exists as a monomer in both the ADP-and ATP-bound forms, as well as in the presence of DNA that contains a single high affinity binding site [9,12], have made it difficult to characterize the exact nature of self-oligomerization.In addition to oligomerization, cross-linking studies have shown that the N-terminal domain of DnaA interacts directly with both DnaB [1] and the regulatory protein DiaA [21]. For DnaB, the regions of interaction were further defined by solid phase binding assays, which showed that residues 24-86 from the N-terminal domain, as well as residues in domain III, were important [20].…”

“…In addition to oligomerization, cross-linking studies have shown that the N-terminal domain of DnaA interacts directly with both DnaB [1] and the regulatory protein DiaA [21]. For DnaB, the regions of interaction were further defined by solid phase binding assays, which showed that residues 24-86 from the N-terminal domain, as well as residues in domain III, were important [20].…”