FIG. 2.

Mapping the regions of Isw2 cross-linked to three sites in the nucleosome by NTCB digestion. (A) A schematic diagram of Isw2 is shown, with residue numbers for the different cysteine cut sites indicated. The locations of the conserved Isw2 domains are indicated, along with the size of each proteolytic fragment (kDa). The set of fragments obtained with single-hit NTCB digestion are shown below the Isw2 schematic, with two fragments created from each single hit depicted as either a shaded or open box. The predicted sets of radiolabeled single-hit proteolytic fragments that can be obtained depending on the site of Isw2 that is cross-linked to DNA are shown to the right. (B) Photoaffinity-labeled Isw2 was digested with the indicated concentrations of NTCB and incubation times, followed by analysis by 4 to 20% Tris-glycine SDS-PAGE. The apparent molecular masses of the radiolabeled bands were calculated as described in Materials and Methods and are shown in kDa. (C) Fragments of Isw2 were synthesized in vitro that correspond to fragments that would be obtained by NTCB digestion (Table 1) and contain either the N terminus (NN1, NN2, NN3, and NN4), the C terminus (NC1, NC2, NC3, and NC4), or an internal fragment (NI2). These truncated proteins were analyzed by 4 to 20% Tris-glycine SDS-PAGE. The molecular masses of the ¹²⁵I-labeled Mark12 protein standards are indicated to the right. The slowest-migrating major bands were fully translated products and were used in calculating the apparent molecular masses.