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Archaea. 2012; 2012: 719092.
Published online 2012 September 3. doi:  10.1155/2012/719092
PMCID: PMC3438722
DNA Binding in High Salt: Analysing the Salt Dependence of Replication Protein A3 from the Halophile Haloferax volcanii
Jody A. Winter,* Bushra Patoli, and Karen A. Bunting
Centre for Genetics and Genomics, University of Nottingham, Queen's Medical Centre, Nottingham NG7 2UH, UK
*Jody A. Winter: jody.winter/at/nottingham.ac.uk
Academic Editor: Yoshizumi Ishino
Received May 9, 2012; Accepted June 18, 2012.
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.
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