|Title||The Escherichia coli GTPase ObgE modulates hydroxyl radical levels in response to DNA replication fork arrest|
|Publication Type||Journal Article|
|Year of Publication||2012|
|Authors||Kint, C. I., N. Verstraeten, I. Wens, V. R. Liebens, J. Hofkens, W. Versées, M. Fauvart, and J. Michiels|
|Date Published||2012 Oct|
|Keywords||Amino Acid Sequence, Chromosomes, Bacterial, DNA Replication, Drug Hypersensitivity, Escherichia coli, Escherichia coli Proteins, Free Radical Scavengers, Gene Deletion, Hydroxyl Radical, Hydroxyurea, Molecular Sequence Data, Monomeric GTP-Binding Proteins, Mutagenesis, Mutation, Nucleic Acid Synthesis Inhibitors, Protein Conformation, Sequence Homology, Amino Acid|
Obg proteins are universally conserved GTP-binding proteins that are essential for viability in bacteria. Homologs in different organisms are involved in various cellular processes, including DNA replication. The goal of this study was to analyse the structure-function relationship of Escherichia coli ObgE with regard to DNA replication in general and sensitivity to stalled replication forks in particular. Defined C-terminal chromosomal deletion mutants of obgE were constructed and tested for sensitivity to the replication inhibitor hydroxyurea. The ObgE C-terminal domain was shown to be dispensable for normal growth of E.coli. However, a region within this domain is involved in the cellular response to replication fork stress. In addition, a mutant obgE over-expression library was constructed by error-prone PCR and screened for increased hydroxyurea sensitivity. ObgE proteins with substitutions L159Q, G163V, P168V, G216A or R237C, located within distinct domains of ObgE, display dominant-negative effects leading to hydroxyurea hypersensitivity when over-expressed. These effects are abolished in strains with a single deletion of the iron transporter TonB or combined deletions the toxin/antitoxin modules RelBE/MazEF, strains both of which have been shown to be involved in a pathway that stimulates hydroxyl radical formation following hydroxyurea treatment. Moreover, the observed dominant-negative effects are lost in the presence of the hydroxyl radical scavenger thiourea. Together, these results indicate involvement of hydroxyl radical toxicity in ObgE-mediated protection against replication fork stress.
|Alternate Journal||FEBS J.|