Modes of overinitiation, dnaA gene expression, and inhibition of cell division in a novel cold-sensitive hda mutant of Escherichia coli.
The chromosomal replication cycle is strictly coordinated with cell cycle progression in Escherichia coli. ATP-DnaA initiates replication, leading to loading of the DNA polymerase III holoenzyme. The DNA-loaded form of the beta clamp subunit of the polymerase binds the Hda protein, which promotes ATP-DnaA hydrolysis, yielding inactive ADP-DnaA. This regulation is required to repress overinitiation. In this study, we have isolated a novel cold-sensitive hda mutant, the hda-185 mutant. The hda-185 mutant caused overinitiation of chromosomal replication at 25 degrees C, which most likely led to blockage of replication fork progress. Consistently, the inhibition of colony formation at 25 degrees C was suppressed by disruption of the diaA gene, an initiation stimulator. Disruption of the seqA gene, an initiation inhibitor, showed synthetic lethality with hda-185 even at 42 degrees C. The cellular ATP-DnaA level was increased in an hda-185-dependent manner. The cellular concentrations of DnaA protein and dnaA mRNA were comparable at 25 degrees C to those in a wild-type hda strain. We also found that multiple copies of the ribonucleotide reductase genes (nrdAB or nrdEF) or dnaB gene repressed overinitiation. The cellular levels of dATP and dCTP were elevated in cells bearing multiple copies of nrdAB. The catalytic site within NrdA was required for multicopy suppression, suggesting the importance of an active form of NrdA or elevated levels of deoxyribonucleotides in inhibition of overinitiation in the hda-185 cells. Cell division in the hda-185 mutant was inhibited at 25 degrees C in a LexA regulon-independent manner, suggesting that overinitiation in the hda-185 mutant induced a unique division inhibition pathway.
Fujimitsu
Kazuyuki
K
Department of Molecular Biology, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan.
Su'etsugu
Masayuki
M
Yamaguchi
Yoko
Y
Mazda
Kensaku
K
Fu
Nisi
N
Kawakami
Hironori
H
Katayama
Tsutomu
T
D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism*; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002352:Carrier Proteins / Q000235:genetics; D002455:Cell Division; D003080:Cold Temperature; D003600:Cytosol / Q000737:chemistry; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D003838:Deoxyadenine Nucleotides / Q000032:analysis; D003845:Deoxycytosine Nucleotides / Q000032:analysis; D053488:DnaB Helicases / Q000235:genetics; D004926:Escherichia coli / Q000166:cytology* / Q000235:genetics / Q000254:growth & development; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D018628:Gene Dosage; D020869:Gene Expression Profiling; D006358:Hot Temperature; D016254:Mutagenesis, Insertional; D009154:Mutation; D012262:Ribonucleoside Diphosphate Reductase / Q000235:genetics; D012264:Ribonucleotide Reductases / Q000235:genetics
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D003838:Deoxyadenine Nucleotides; D003845:Deoxycytosine Nucleotides; C523634:DiaA protein, E coli; C066312:DnaA protein, Bacteria; D029968:Escherichia coli Proteins; C089746:SeqA protein, E coli; C024107:2'-deoxycytidine 5'-triphosphate; C114872:NrdF protein, bacteria; D012264:Ribonucleotide Reductases; C449642:NrdA protein, E coli; D012262:Ribonucleoside Diphosphate Reductase; D000251:Adenosine Triphosphatases; C435211:Hda protein, E coli; C506730:dnaB protein, E coli; D053488:DnaB Helicases; C026600:2'-deoxyadenosine triphosphate