DNA replication-coupled inactivation of DnaA protein in vitro: a role for DnaA arginine-334 of the AAA+ Box VIII motif in ATP hydrolysis.
The DnaA protein, which initiates chromosomal replication in Escherichia coli, is negatively regulated by both the sliding clamp of DNA polymerase III holoenzyme and the IdaB protein. We have found that, when the amount of minichromosome is limited in an in vitro replication system, minichromosomal replication-stimulated hydrolysis of DnaA-bound ATP yields the ADP-bound inactive form. The number of sliding clamps formed during replication was at least five per minichromosome, which is 2.7-fold higher than the number formed during incubation without replication. These results support the notion that coupling of DnaA-ATP hydrolysis to DNA replication is the outcome of enhanced clamp formation. We have also found that the amino acid substitution R334H in DnaA severely inhibits the hydrolysis of bound ATP in vitro. Whereas ATP bound to wild-type DnaA is hydrolysed in a DNA-dependent intrinsic manner or in a sliding clamp-dependent manner, ATP bound to DnaA R334H protein was resistant to hydrolysis under the same conditions. This arginine residue may be located in the vicinity where ATP binds, and therefore may play an essential role in ATP hydrolysis. This residue is highly conserved among DnaA homologues and also in the Box VIII motif of the AAA+ protein family.
Su'etsugu
M
M
Department of Molecular Microbiology, Kyushu University Graduate School of Pharmaceutical Sciences, Fukuoka 812-8582, Japan.
Kawakami
H
H
Kurokawa
K
K
Kubota
T
T
Takata
M
M
Katayama
T
T
D000255:Adenosine Triphosphate / Q000378:metabolism*; D020816:Amino Acid Motifs; D001120:Arginine / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004261:DNA Replication; D004269:DNA, Bacterial / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D006868:Hydrolysis; D018741:Replication Origin
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; C066312:DnaA protein, Bacteria; D000255:Adenosine Triphosphate; D001120:Arginine