FIG. 3.

Cse4 association is specific to STB-containing DNA. (A) Schematic diagram of the rationale of the analysis. If Cse4 association is specific to a plasmid locale, DNA digestion with the restriction enzymes RE1 and RE2 should release the fragment lacking Cse4 from its association with the Cse4-directed antibody. (B and C) The STB (B) or CEN (C) reporter plasmid employed in the assays is schematically diagrammed at the top. Restriction enzyme digestions were performed on protein A-Dynabeads, and DNAs in the released or retained fractions were analyzed by gel electrophoresis and Southern hybridization using the indicated probes. For reference, enzyme digestions performed on total DNA (“naked” DNA) prepared from the experimental strains were analyzed similarly. Hybridization profiles from the relevant portions of the gels are displayed. RI, HIII, and BII stand for EcoRI, HindIII, and BglII, respectively. In, input; U, unbound; S, supernatant; W, wash; SC, supercoiled plasmid.