The interaction of DiaA and DnaA regulates the replication cycle in E. coli by directly promoting ATP-DnaA-specific initiation complexes

In prokaryotes and eukaryotes, the initiation of chromosomal replication is strictly regulated and occurs at a specific time during the cell cycle (Messer 2002; Stillman 2005; Kaguni 2006; Botchan 2007). A crucial aspect of replication initiation is the ordered assembly and conformational change of specific proteins at the replication origin. In Escherichia coli, multiple ATP-bound DnaA (ATP–DnaA) molecules form active complexes with the origin of replication (oriC) and unwind the duplex DNA within oriC, resulting in replication initiation. DnaA monomers specifically bind to sequences within oriC called DnaA boxes and interact with other DnaA molecules. These activities potentiate the formation of DnaA homomultimers on oriC. The present study is the first demonstration that DiaA, a DnaA-binding protein (Ishida et al. 2004), forms homotetramers, directly stimulates assembly of multiple ATP–DnaA molecules on oriC, and positively regulates a conformational change of the DnaA multimer–oriC complex to an initiation-competent state. A single E. coli cell contains ∼1000 molecules of the initiator protein DnaA, which exists in either an ATP–DnaA active form or an ADP-bound inactive form (ADP–DnaA) (Messer 2002; Kaguni 2006). The level of ATP–DnaA in the cell temporarily increases during the cell cycle at the same time as the initiation of replication (Kurokawa et al. 1999). The E. coli oriC contains several DnaA-binding sites, including several repeats of a 9-mer sequence called the DnaA box. When ATP–DnaA forms a multimeric complex on oriC, the DNA duplex is unwound specifically at 13-mer AT-rich repeats within oriC (Speck and Messer 2001; McGarry et al. 2004; Kawakami et al. 2005). DnaB helicase is then loaded onto the unwound strands of DNA, and the single-stranded region is extended. DnaG primase and DNA polymerase (Pol) III holoenzyme then assemble on the single-stranded DNA to replicate the complementary strand. After the completion of Okazaki fragments, the β-clamp, which is a component of Pol III holoenzyme, remains on the nascent DNA strand (O’Donnell 2006). Hda forms a complex with the DNA-loaded β-clamp, which promotes hydrolysis of DnaA–ATP to ADP–DnaA (Katayama et al. 1998; Kato and Katayama 2001). This process for inactivating DnaA, termed the regulatory inactivation of DnaA (RIDA), is required for repressing untimely or excessive initiation events. The timing of replication initiation is tightly coupled with cell cycle progression (Zyskind and Smith 1992). In E. coli, when the growth rate of cells is rapid, a single cell contains replication intermediates on multiple chromosomes, and replication initiates on each chromosome at exactly the same time. This level of regulation implies that there are multiple mechanisms in place to ensure that replication initiation is coupled to specific cell cycle events, that ATP–DnaA molecules accumulate to the proper level, and that the formation of ATP–DnaA–oriC complexes occurs in a timely manner. These mechanisms of regulation have yet to be fully explored. In eukaryotes, initiation of DNA replication requires many proteins that directly or indirectly interact with the replication origin (Stillman 2005; Botchan 2007). In Saccharomyces cerevisiae, the origin recognition complex (ORC), a nucleoprotein complex that binds to the origin of replication, is preserved during the cell cycle. A pre-replication complex is formed when the MCM helicase complex is loaded onto the ORC–DNA complex, which also contains Cdc6 and Cdt1. The initiation of replication is activated by cyclin-dependent kinase at the G1/S transition, and several proteins, including Cdc45, the 11–3–2 complex, and the GINS complex are loaded onto the pre-replication complex to form an initiation-competent replication complex in a timely manner. In E. coli, DiaA is required to ensure the timely initiation of chromosomal replication during the cell cycle (Ishida et al. 2004). The diaA gene was first identified in a search for intergenic suppressor mutations of the dnaA mutant dnaAcos, in which there is overinitiation of chromosomal replication, and colony formation at 30°C is inhibited. Replication initiation in the dnaAcos mutant is resistant to RIDA at this temperature (Katayama and Crooke 1995). In diaA gene-disrupted cells, initiation is retarded, and the synchronous initiation of replication on multiple chromosomes at oriC is disrupted (Ishida et al. 2004). In the diaA mutant, when two oriC copies are present in a single, rapidly growing cell, only one oriC initiates, which produces a cell bearing three copies of oriC. In wild-type cells, this is an extremely rare event. Mutation of diaA also inhibits the stable maintenance of minichromosomes and, in a dnaA46-temperature sensitive mutant, causes synthetic lethality at 37°C. Thus, DiaA is a positive regulator to stimulate the initiation. This function of DiaA is independent of RIDA and SeqA, an oriC-binding protein that represses extra initiations (Slater et al. 1995). Purified DiaA directly and specifically binds to DnaA, independent of the nucleotide- or DNA-bound state of DnaA (Ishida et al. 2004). DiaA binds to domains I–II in the N terminus of DnaA. The overall primary sequence of DiaA indicates that it contains a sugar isomerase (SIS) domain (Bateman 1999). This domain is proposed to be a consensus site for binding phosphosugars and is found in several proteins, such as the glucokinase regulator protein. In the present study, we isolated several plasmid-encoded dysfunctional diaA mutants using complementation analysis of the dnaAcos diaA∷Tn5 mutant. In parallel, we determined the crystal structure of DiaA at 1.8 A resolution. The structure revealed that DiaA forms a homotetramer consisting of a symmetrical pair of homodimers. Analysis of mutant DiaA proteins identified residues specifically required for DnaA binding. We also identified specific sites located in the interdimer interface that were required for homotetramer formation. Another group of mutations was located in the putative phosphosugar-binding region. All the DiaA mutants were inactive in stimulating replication initiation. We also showed that a single DiaA tetramer can bind multiple DnaA molecules simultaneously. DiaA tetramers stimulated the assembly of multiple DnaA molecules on oriC, ATP–DnaA-specific conformational changes of the initiation complex, and unwinding of the duplex DNA at oriC. Mutant DiaA proteins were defective in all these activities. We propose that DiaA regulates initiation via a novel mechanism, in which a single DiaA tetramer binds to multiple DnaA molecules to stimulate inter-DnaA interactions, resulting in efficient assembly of multiple ATP–DnaA molecules on oriC and stimulation of initiation complex formation. This unique role for DiaA is most likely essential for the promotion of initiation in a cell cycle coordinated manner.