Fig 1.
In vitro activity of Rtt109-Vps75 on linker histone H1. (A) Rtt109-Vps75 but not Rtt109-Asf1 acetylates vertebrate linker histone. HAT assays were carried out with [14C]acetyl-CoA before being resolved by 15% SDS-PAGE. Gels were either transferred onto nitrocellulose and immunoblotted with anti-HIS, anti-H3K56ac, or anti-H3K9ac or stained with Coomassie blue (CB), saturated with Enlightning (PerkinElmer), and dried under vacuum, and then 14C was imaged using a Typhoon phosphorimager. The asterisk indicates a breakdown product of 6×HIS-Vps75. (B) Increasing concentrations of Vps75 correlate with increasing acetylation activity of rRtt109 on bovine linker histone. Increasing concentrations of either 6×HIS-Vps75 or 6×HIS-Asf1 were incubated with a constant amount of full-length 6×HIS-Rtt109 and bovine H1. HAT assays were carried out and analyzed by SDS-PAGE/autoradiography as described for panel A. The asterisk indicates a probable dimeric form of 6×HIS-Rtt109. (C) Rtt109-Vps75 acetylates Hho1 in vitro. Recombinant proteins were incubated with either chicken core histones or 6×HIS-Hho1 as indicated. In vitro HAT assay reactions were carried out and analyzed as described for panel A. The asterisk indicates a probable dimeric form of 6×HIS-Rtt109. α, anti.