title: ssb gene duplication restores the viability of ΔholC and ΔholD Escherichia coli mutants.
abstract: The HolC-HolD (χψ) complex is part of the DNA polymerase III holoenzyme (Pol III HE) clamp-loader. Several lines of evidence indicate that both leading- and lagging-strand synthesis are affected in the absence of this complex. The Escherichia coli ΔholD mutant grows poorly and suppressor mutations that restore growth appear spontaneously. Here we show that duplication of the ssb gene, encoding the single-stranded DNA binding protein (SSB), restores ΔholD mutant growth at all temperatures on both minimal and rich medium. RecFOR-dependent SOS induction, previously shown to occur in the ΔholD mutant, is unaffected by ssb gene duplication, suggesting that lagging-strand synthesis remains perturbed. The C-terminal SSB disordered tail, which interacts with several E. coli repair, recombination and replication proteins, must be intact in both copies of the gene in order to restore normal growth. This suggests that SSB-mediated ΔholD suppression involves interaction with one or more partner proteins. ssb gene duplication also suppresses ΔholC single mutant and ΔholC ΔholD double mutant growth defects, indicating that it bypasses the need for the entire χψ complex. We propose that doubling the amount of SSB stabilizes HolCD-less Pol III HE DNA binding through interactions between SSB and a replisome component, possibly DnaE. Given that SSB binds DNA in vitro via different binding modes depending on experimental conditions, including SSB protein concentration and SSB interactions with partner proteins, our results support the idea that controlling the balance between SSB binding modes is critical for DNA Pol III HE stability in vivo, with important implications for DNA replication and genome stability.
authors.affiliation: Centre de Génétique Moléculaire, Centre National de la Recherche Scientifique, Gif-sur-Yvette, France.
authors.forename: Stéphane
authors.initials: S
authors.lastname: Duigou
authors.affiliation: Plateforme Intégrée IMAGIF, Centre National de la Recherche Scientifique, Gif-sur-Yvette, France.
authors.forename: Maud
authors.initials: M
authors.lastname: Silvain
authors.affiliation: Área de Genética, Facultad de Ciencias, Universidad de Málaga, Málaga, Spain.
authors.forename: Enrique
authors.initials: E
authors.lastname: Viguera
authors.affiliation: Centre de Génétique Moléculaire, Centre National de la Recherche Scientifique, Gif-sur-Yvette, France.
authors.forename: Bénédicte
authors.initials: B
authors.lastname: Michel
mesh_terms: D004258:DNA Polymerase III / Q000235:genetics* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism; D020440:Gene Duplication; D015964:Gene Expression Regulation, Bacterial; D009154:Mutation; D013014:SOS Response, Genetics; D013489:Suppression, Genetic; D013696:Temperature
chemical_list: D004268:DNA-Binding Proteins; C094399:DinB protein, E coli; D029968:Escherichia coli Proteins; C476430:SSB protein, E coli; C459959:holD protein, E coli; D004258:DNA Polymerase III; C476431:holC protein, E coli