Mutant DnaA proteins defective in duplex opening of oriC, the origin of chromosomal DNA replication in Escherichia coli.
We characterized three mutant DnaA proteins with an amino acid substitution of R334H, R342H and E361G that renders chromosomal replication cold (20 degrees C) sensitive. Each mutant DnaA protein was highly purified from overproducers, and replication activities were assayed in in vitro oriC replication systems. At 30 degrees C, all three mutant proteins exhibited specific activity similar to that seen with the wild-type protein, whereas at 20 degrees C, there was much less activity in a replication system using a crude replicative extract. Regarding the affinity for ATP, the dissociation rate of bound ATP and binding to oriC DNA, the three mutant DnaA proteins showed a capacity indistinguishable from that of the wild-type DnaA protein. Activity for oriC DNA unwinding of the two mutant DnaA proteins, R334H and R342H, was more sensitive to low temperature than that of the wild-type DnaA protein. We propose that R334H and R342H have a defect in their potential to unwind oriC DNA at low temperatures, the result being the cold-sensitive phenotype in oriC DNA replication. The two amino acid residues of DnaA protein, located in a motif homologous to that of NtrC protein, may play a role in the formation of the open complex. The E361 residue may be related to interaction with another protein present in a crude cell extract.
Takata
M
M
Department of Molecular Microbiology, Kyushu University Graduate School of Pharmaceutical Sciences, Maidashi 3-1-1, Higashi-ku, Fukuoka 812-8582, Japan.
Guo
L
L
Katayama
T
T
Hase
M
M
Seyama
Y
Y
Miki
T
T
Sekimizu
K
K
D000255:Adenosine Triphosphate / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003080:Cold Temperature; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development; D009154:Mutation; D051738:Origin Recognition Complex; D015719:Single-Strand Specific DNA and RNA Endonucleases / Q000378:metabolism; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; C066312:DnaA protein, Bacteria; C114650:OriC chromosomal replication origin; D051738:Origin Recognition Complex; D014764:Viral Proteins; D000255:Adenosine Triphosphate; D015719:Single-Strand Specific DNA and RNA Endonucleases