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Title: The Escherichia coli GTPase ObgE modulates hydroxyl radical levels in response to DNA replication fork arrest
Authors: Kint, Cyrielle
Verstraeten, Natalie
Wens, I.
Liebens, Veerle
Hofkens, Johan
Versées, W.
Fauvart, Maarten
Michiels, Jan # ×
Issue Date: Oct-2012
Publisher: Published by Blackwell Pub. on behalf of the Federation of European Biochemical Societies
Series Title: FEBS Journal vol:279 issue:19 pages:3692-3704
Abstract: 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.
URI: 
ISSN: 1742-464X
Publication status: published
KU Leuven publication type: IT
Appears in Collections:Centre of Microbial and Plant Genetics
Molecular Imaging and Photonics
× corresponding author
# (joint) last author

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