Rapid exchange of bound ADP on the Staphylococcus aureus replication initiation protein DnaA.
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 ATP-DnaA to ADP-DnaA in S. aureus and thereby delaying the re-initiation of DNA replication.
Laboratory of Microbiology, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo 113-0033, Japan. kurokawa@mol.f.u-tokyo.ac.jp
Kenji
K
Kurokawa
Hikaru
H
Mizumura
Tohru
T
Takaki
Yumiko
Y
Ishii
Norikazu
N
Ichihashi
Bok Luel
BL
Lee
Kazuhisa
K
Sekimizu
D000244:Adenosine Diphosphate / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry; D001426:Bacterial Proteins / Q000378:metabolism*; D004261:DNA Replication; D004269:DNA, Bacterial / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D006868:Hydrolysis; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D013211:Staphylococcus aureus / Q000378:metabolism*; D013297:Streptococcus pyogenes / Q000378:metabolism; D013997:Time Factors
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; C066312:DnaA protein, Bacteria; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate