Figure 4.

The amino acids between position 47 and 74 are important for Ref nuclease activity on circular ssDNA. (A) Schematic of Ref circular ssDNA nuclease assay, as described in Materials and Methods. (B) Agarose gel of reactions after 20-min incubation of circular ssDNA with all Ref variants. All circular ssDNA is degraded within 20 min with full-length WT ϕW39, ϕW39 ΔN21, ϕW39 ΔN47 and ϕW39 ΔN59 Ref. The ϕW39 ΔN66 and ϕW39 ΔN74 showed reduced activity at similar concentrations, but regained WT activity level with a 10-fold increase in protein concentration.