title: DNA binding in high salt: analysing the salt dependence of replication protein A3 from the halophile Haloferax volcanii.
abstract: Halophilic archaea maintain intracellular salt concentrations close to saturation to survive in high-salt environments and their cellular processes have adapted to function under these conditions. Little is known regarding halophilic adaptation of the DNA processing machinery, particularly intriguing since protein-DNA interactions are classically salt sensitive. To investigate such adaptation, we characterised the DNA-binding capabilities of recombinant RPA3 from Haloferax volcanii (HvRPA3). Under physiological salt conditions (3 M KCl), HvRPA3 is monomeric, binding 18 nucleotide ssDNA with nanomolar affinity, demonstrating that RPAs containing the single OB-fold/zinc finger architecture bind with broadly comparable affinity to two OB-fold/zinc finger RPAs. Reducing the salt concentration to 1 M KCl induces dimerisation of the protein, which retains its ability to bind DNA. On circular ssDNA, two concentration-dependent binding modes are observed. Conventionally, increased salt concentration adversely affects DNA binding but HvRPA3 does not bind DNA in 0.2 M KCl, although multimerisation may occlude the binding site. The single N-terminal OB-fold is competent to bind DNA in the absence of the C-terminal zinc finger, albeit with reduced affinity. This study represents the first quantitative characterisation of DNA binding in a halophilic protein in extreme salt concentrations.
authors.affiliation: Centre for Genetics and Genomics, University of Nottingham, Queen's Medical Centre, Nottingham NG7 2UH, UK. jody.winter@nottingham.ac.uk
authors.forename: Jody A
authors.initials: JA
authors.lastname: Winter
authors.forename: Bushra
authors.initials: B
authors.lastname: Patoli
authors.forename: Karen A
authors.initials: KA
authors.lastname: Bunting
mesh_terms: D000220:Adaptation, Biological; D019843:Archaeal Proteins / Q000737:chemistry* / Q000235:genetics; D001665:Binding Sites; D002850:Chromatography, Gel / Q000379:methods; D004261:DNA Replication; D019641:DNA, Archaeal / Q000737:chemistry*; D004277:DNA, Single-Stranded / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D019622:Haloferax volcanii / Q000737:chemistry* / Q000235:genetics; D011189:Potassium Chloride / Q000737:chemistry; D055503:Protein Multimerization; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D013045:Species Specificity; D014131:Trace Elements / Q000737:chemistry; D016335:Zinc Fingers
chemical_list: D019843:Archaeal Proteins; D019641:DNA, Archaeal; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D014131:Trace Elements; D011189:Potassium Chloride