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

Winter, J.A. ORCID: 0000-0003-3582-7596, Patoli, B. and Bunting, K.A., 2012. DNA Binding in High Salt: Analysing the Salt Dependence of Replication Protein A3 from the Halophile Haloferax volcanii. Archaea, 2012 (719092), pp. 1-12. ISSN 1472-3646

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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 (3M 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 1M 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 0.2M 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.

Item Type: Journal article
Publication Title: Archaea
Creators: Winter, J.A., Patoli, B. and Bunting, K.A.
Publisher: Hindawi Publishing Corporation
Date: 2012
Volume: 2012
Number: 719092
ISSN: 1472-3646
Identifiers:
NumberType
10.1155/2012/719092DOI
Divisions: Schools > School of Science and Technology
Depositing User: Linda Sullivan
Date Added: 03 Nov 2015 16:27
Last Modified: 09 Jun 2017 13:56
URI: http://irep.ntu.ac.uk/id/eprint/26136

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