Modes of Overinitiation, dnaA Gene Expression, and Inhibition of Cell Division in a Novel Cold-Sensitive hda Mutant of Escherichia coli

Fujimitsu, K., Su'etsugu, M., Yamaguchi, Y., Mazda, K., Fu, Nisi, Kawakami, H., Katayama, T. (August 2008) Modes of Overinitiation, dnaA Gene Expression, and Inhibition of Cell Division in a Novel Cold-Sensitive hda Mutant of Escherichia coli. J. Bacteriol., 190 (15). pp. 5368-5381.

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URL: https://www.ncbi.nlm.nih.gov/pubmed/18502852
DOI: 10.1128/JB.00044-08

Abstract

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.

Item Type: Paper
Subjects: bioinformatics > genomics and proteomics > analysis and processing > DNA RNA Processing
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification
organism description > bacteria
CSHL Authors:
Communities: CSHL labs > Stillman lab
Depositing User: Tom Adams
Date: 1 August 2008
Date Deposited: 12 Jul 2011 18:34
Last Modified: 14 Mar 2018 16:23
PMCID: PMC2493284
Related URLs:
URI: http://repository.cshl.edu/id/eprint/7737

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