DNA Binding in High Salt : Analysing the Salt Dependence of Replication Protein A3 from the Halophile Haloferax volcanii

Abstract EN

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.