FIG. 5.
Binding of wild-type or mutant AR1 to ssDNA-cellulose: SDS-PAGE of AR1 (wild-type [wt] and mutants as indicated) bound to ssDNA-cellulose and eluted at the salt concentrations indicated. [35S]methionine-labeled in vitro-synthesized AR1 was incubated with ssDNA coupled to cellulose resin. Protein was eluted from resins by successive washes with buffer containing increasing concentrations of KCl up to 1 M. Following the 1 M salt elution, no residual bound protein was eluted by boiling in SDS sample buffer (not shown). Equivalent amounts of each eluted fraction, including the LOAD (input protein), FT (flowthrough protein not bound to resin), and WASH (final wash of resin with loading buffer prior to elutions with higher salt) lanes, were analyzed by SDS-PAGE. Not shown were AR1Q74A/R75A/H76A, AR1K122A/K124A, and AR1K213A/Y214A/E215A, all of which retained the ability to mask and had essentially the same profile of ssDNA binding as wild-type AR1 and AR1K164A/N165A/D166A. See text for details.