TY - JOUR
T1 - Stable nucleotide binding to DnaA requires a specific glutamic acid residue within the AAA+ box II motif
AU - Ozaki, Shogo
AU - Noguchi, Yasunori
AU - Nishimura, Masahiro
AU - Katayama, Tsutomu
N1 - Funding Information:
We are grateful to Chika Odate for plasmid construction. This study was supported by Grant-in-aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
PY - 2012/8
Y1 - 2012/8
N2 - In complex with ATP, but not ADP, DnaA protein multimers unwind a specific region of duplex DNA within the chromosomal replication origin, oriC, triggering a series of reactions that result in initiation of DNA replication. Following replication initiation, ATP hydrolysis, which is coupled to DNA replication, results in the generation of initiation-incompetent ADP-DnaA. Suppression of overinitiation of replication requires that ADP-DnaA complexes be stably maintained until the next round of replication. Thus, the functional and structural requirements that ensure stable nucleotide binding to DnaA are crucial for proper regulation of replication. Here, we demonstrate that Glu143 of DnaA, located within the AAA+ box II N-linker motif, is a key residue involved in stable nucleotide binding. A Glu143 substitution variant of DnaA (DnaA E143A) bound to ADP on ice with an affinity similar to wild-type DnaA, but the resultant ADP-DnaA E143A complex was more labile at 37. °C than wild-type ADP-DnaA complexes. Consistent with this, conversion of ADP-DnaA E143A to ATP-DnaA E143A was stimulated at 37. °C in the presence of ATP, which also stimulated replication of a minichromosome in an in vitro reconstitution reaction. Expression of DnaA E143A in vivo inhibited cell growth in an oriC-dependent manner, suggesting that DnaA E143A caused over-initiation of replication, consistent with the in vitro results. Glu is a highly conserved residue at the corresponding position of γ-proteobacterial DnaA orthologs. Our finding of the novel role for the DnaA N-linker region may represent a conserved function of this motif among those DnaA orthologs.
AB - In complex with ATP, but not ADP, DnaA protein multimers unwind a specific region of duplex DNA within the chromosomal replication origin, oriC, triggering a series of reactions that result in initiation of DNA replication. Following replication initiation, ATP hydrolysis, which is coupled to DNA replication, results in the generation of initiation-incompetent ADP-DnaA. Suppression of overinitiation of replication requires that ADP-DnaA complexes be stably maintained until the next round of replication. Thus, the functional and structural requirements that ensure stable nucleotide binding to DnaA are crucial for proper regulation of replication. Here, we demonstrate that Glu143 of DnaA, located within the AAA+ box II N-linker motif, is a key residue involved in stable nucleotide binding. A Glu143 substitution variant of DnaA (DnaA E143A) bound to ADP on ice with an affinity similar to wild-type DnaA, but the resultant ADP-DnaA E143A complex was more labile at 37. °C than wild-type ADP-DnaA complexes. Consistent with this, conversion of ADP-DnaA E143A to ATP-DnaA E143A was stimulated at 37. °C in the presence of ATP, which also stimulated replication of a minichromosome in an in vitro reconstitution reaction. Expression of DnaA E143A in vivo inhibited cell growth in an oriC-dependent manner, suggesting that DnaA E143A caused over-initiation of replication, consistent with the in vitro results. Glu is a highly conserved residue at the corresponding position of γ-proteobacterial DnaA orthologs. Our finding of the novel role for the DnaA N-linker region may represent a conserved function of this motif among those DnaA orthologs.
UR - http://www.scopus.com/inward/record.url?scp=84864385103&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84864385103&partnerID=8YFLogxK
U2 - 10.1016/j.jsb.2012.05.001
DO - 10.1016/j.jsb.2012.05.001
M3 - Article
C2 - 22579783
AN - SCOPUS:84864385103
VL - 179
SP - 242
EP - 250
JO - Journal of Structural Biology
JF - Journal of Structural Biology
SN - 1047-8477
IS - 2
ER -