Figure 5

Biochemical analysis of Asf1 residues important for histone binding. (A) Asf1 proteins containing mutations in the H3 binding surface fail to pull-down histones H3/H4. Extracts were made from E. coli cells expressing yeast histones H3 and H4, as well as either wild type or mutant His6-tagged Asf1. Talon metal affinity resin was used to precipitate Asf1 and associated proteins. Samples were separated on a 17% SDS-PAGE gel, visualized by Coomassie staining to indicate recovery of Asf1 (upper panel), or analyzed by Western blotting using an anti-H3 antibody (AbCam) (lower panel). Sc H3/H4 indicates recombinant yeast histones H3/H4 (0.5 μg upper gel; 10 ng lower gel). (B) Mutation of Asf1 residues outside of the histone-binding patch does not disrupt its interaction with histones H3/H4. The experiment was performed as above.