Rapid Exchange of Bound ADP on the Staphylococcus aureus Replication Initiation Protein DnaA

Abstract

In Escherichia coli, regulatory inactivation of the replication initiator DnaA occurs after initiation as a result of hydrolysis of bound ATP to ADP, but it has been unknown how DnaA is controlled to coordinate cell growth and chromosomal replication in Gram-positive bacteria such as Staphylococcus aureus. This study examined the roles of ATP binding and its hydrolysis in the regulation of the S. aureus DnaA activity. In vitro, S. aureus DnaA melted S. aureus oriC in the presence of ATP but not ADP by a mechanism independent of ATP hydrolysis. Unlike E. coli DnaA, binding of ADP to S. aureus DnaA was unstable. As a result, at physiological concentrations of ATP, ADP bound to S. aureus DnaA was rapidly exchanged for ATP, thereby regenerating the ability of DnaA to form the open complex in vitro. Therefore, we examined whether formation of ADP-DnaA participates in suppression of replication initiation in vivo. Induction of the R318H mutant of the AAA؉ sensor 2 protein, which has decreased intrinsic ATPase activity, caused over-initiation of chromosome replication in S. aureus, suggesting that formation of ADP-DnaA suppresses the initiation step in S. aureus. Together with the biochemical features of S. aureus DnaA, the weak ability to convert ATP-DnaA into ADP-DnaA and the instability of ADP-DnaA, these results suggest that there may be unidentified system(s) for reducing the cellular ratio of ATPDnaA to ADP-DnaA in S. aureus and thereby delaying the reinitiation of DNA replication.The initiation step of chromosome replication is an important set point for coordinating cell growth and chromosomal replication, and it is tightly regulated so that it takes place only once per cell cycle. DNA replication in bacteria starts when the cell mass reaches an appropriate size and at the appropriate time, which is controlled in part by the quantity and activity of initiator DnaA protein (1-3). In Escherichia coli, DnaA binds to 9-mer DnaA boxes in oriC, the origin of chromosome replication, and melts the duplex at AT-rich 13-mers adjacent to oriC. DnaA then directs loading of the DnaB replicative helicase, which depends on the DnaC helicase loader and priming by the DnaG primase. This is followed by the synthesis of DNA by the DNA polymerase III holoenzyme (4).DnaA is composed of four domains: N-terminal domains I and II are involved in DnaA oligomerization and DnaADnaB interaction, and they show a diversity of sequence among bacteria. Domain III contains AAAϩ motifs, and the C-terminal domain IV mediates DNA binding (3, 5). Biochemical and genetic studies in E. coli have revealed adenine nucleotide binding-mediated control of the DnaA activity (6 -11). Specifically, ATP-bound DnaA actively promotes replication initiation, whereas the ADP-bound form is inert (6, 7). ATP promotes the self-assembly of DnaA at replication origins via a conformational change to form a righthanded helical filament (8, 9). In E. coli, 70% to 80% of DnaA exists as the inactive ADP-bound form, and active ATPDnaA levels increase during the initiation ...

The Primosomal Protein DnaD Inhibits Cooperative DNA Binding by the Replication Initiator DnaA in Bacillus subtilis

“…Upon addition of DNA, the rate of hydrolysis increased approximately 6-fold to 12 mol of ATP hydrolyzed per mole of DnaA per hour. These rates of ATP hydrolysis and the effects of DNA are consistent with previously published data for DnaA from E. coli and S. aureus (32,57). In contrast to the wild-type protein, DnaA (K157A) and DnaA(D215A) had rates of ATP hydrolysis of approximately 0.07 and 0.1 mol of ATP per mole of DnaA per hour, respectively ( Table 2).…”

“…Based on these results, we conclude that the rate of nucleotide exchange for B. subtilis DnaA is relatively high compared to the 45-min half-life of exchange for E. coli DnaA (57) and that DnaA-ATP is regenerated from DnaA-ADP in the absence of any other cellular factors. The relatively high rate of nucleotide exchange is similar to that of DnaA from S. aureus (32).…”

“…We found that the rate of nucleotide exchange for B. subtilis DnaA was relatively high, similar to that of DnaA from Staphylococcus aureus (32) and in contrast to the low rate of exchange for DnaA from E. coli (57). DnaD had no effect on the rate of exchange of B. subtilis DnaA.…”

“…Nucleotide exchange was measured using [␣- 32 P]ATP or [ 14 C]ADP. Exchange reaction mixtures contained 40 mM HEPES, pH 7.5, 10 mM magnesium acetate, 0.5 mM EDTA, 1 mM DTT, 150 mM potassium glutamate, 100 g/ml bovine serum albumin (BSA), 10% glycerol, and 1 M [␣-32 P]ATP or 1 M [ 14 C]ADP in the absence or presence of DnaD (600 nM).…”

Stable nucleotide binding to DnaA requires a specific glutamic acid residue within the AAA+ box II motif

“…1B). The stability of the nucleotide-bound forms of these DnaA orthologs is reportedly reduced in vitro, although nucleotide binding to S. aureus DnaA is believed to be stabilized in vivo by an as-yet unknown mechanism of regulation of replication initiation (Kurokawa et al, 2009). A hydrocarbon chain on the second residue therefore might be important for stable nucleotide binding to DnaA orthologs in vivo.…”