title: Insufficient levels of the nrdAB-encoded ribonucleotide reductase underlie the severe growth defect of the Δhda E. coli strain.
abstract: The ATP-bound form of the Escherichia coli DnaA replication initiator protein remodels the chromosomal origin of replication, oriC, to load the replicative helicase. The primary mechanism for regulating the activity of DnaA involves the Hda and β clamp proteins, which act together to dramatically stimulate the intrinsic DNA-dependent ATPase activity of DnaA via a process termed Regulatory Inactivation of DnaA. In addition to hyperinitiation, strains lacking hda function also exhibit cold sensitive growth at 30°C. Strains impaired for the other regulators of initiation (i.e., ΔseqA or ΔdatA) fail to exhibit cold sensitivity. The goal of this study was to gain insight into why loss of hda function impedes growth. We used a genetic approach to isolate 9 suppressors of Δhda cold sensitivity, and characterized the mechanistic basis by which these suppressors alleviated Δhda cold sensitivity. Taken together, our results provide strong support for the view that the fundamental defect associated with Δhda is diminished levels of DNA precursors, particularly dGTP and dATP. We discuss possible mechanisms by which the suppressors identified here may regulate dNTP pool size, as well as similarities in phenotypes between the Δhda strain and hda+strains exposed to the ribonucleotide reductase inhibitor hydroxyurea.
authors.affiliation: Department of Biochemistry, Jacobs School of Medicine and Biomedical Sciences, University at Buffalo, State University of New York, Buffalo, NY, USA.
authors.forename: Vignesh M P
authors.initials: VMP
authors.lastname: Babu
authors.affiliation: Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, Research Triangle Park, NC, USA.
authors.forename: Mark
authors.initials: M
authors.lastname: Itsko
authors.affiliation: Department of Biochemistry, Jacobs School of Medicine and Biomedical Sciences, University at Buffalo, State University of New York, Buffalo, NY, USA.
authors.forename: Jamie C
authors.initials: JC
authors.lastname: Baxter
authors.affiliation: Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, Research Triangle Park, NC, USA.
authors.forename: Roel M
authors.initials: RM
authors.lastname: Schaaper
authors.affiliation: Department of Biochemistry, Jacobs School of Medicine and Biomedical Sciences, University at Buffalo, State University of New York, Buffalo, NY, USA.
authors.forename: Mark D
authors.identifier: http://orcid.org/0000-0002-8221-9297
authors.initials: MD
authors.lastname: Sutton
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism; D000483:Alleles; D003080:Cold Temperature; D004265:DNA Helicases / Q000235:genetics / Q000378:metabolism; D004261:DNA Replication; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D003838:Deoxyadenine Nucleotides / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism*; D012262:Ribonucleoside Diphosphate Reductase / Q000235:genetics* / Q000378:metabolism*; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism
chemical_list: D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D003838:Deoxyadenine Nucleotides; D029968:Escherichia coli Proteins; D015534:Trans-Activators; C045264:replication initiator protein; C449642:NrdA protein, E coli; D012262:Ribonucleoside Diphosphate Reductase; D000251:Adenosine Triphosphatases; D004265:DNA Helicases; C026600:2'-deoxyadenosine triphosphate