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

Figure 2

(a) Size exclusion profiles of HvRPA3 in the presence (dashed line) and absence (solid line) of equimolar (based on monomeric protein concentration) 18mer ssDNA in 0.2, 1, and 3 M KCl. The 𝑥 axis shows elution volume (mls) and the 𝑦 axis absorbance at 280 nm. Arrows indicate the elution position of ssDNA. (b) Agarose gel retardation assay of HvRPA3 titrated into reactions containing PhiX174 ssDNA. −ve indicates negative control containing no HvRPA3. Increasing quantities of HvRPA3 were used −11, 22, 33, 55, 75, 98, 120, and 125 μg. Asterisks indicate the concentration-dependent, differentially migrating forms of complex. (c) Titration of the indicated concentrations of HvRPA3 (calculated for monomer due to the potential independence of binding sites in the dimeric form) plotted against normalised FA of the Cy5-labelled 18mer in 1 M (solid line) and 3 M (dashed line) KCl. Data were fitted to a Hill binding model. Produced using GraphPad Prism.