Figure 8
Effects of H4 tail mutations on histone octamer and nucleosome formation. Size exclusion chromatography (SEC) profile of histone octamer species shows that H4G94P and H4∆94 fail to form octamers. The wild-type (WT) elution positions of each species of WT H3/H4 octamer, H3/H4 tetramer, and H2A/H2B dimers are shown as thin lines. The samples containing H4G94P (dark line) and H4∆94 (dotted line), prepared identically to the WT octamer, elute at the tetramer and dimer elution volumes. (B) H4G94P and H4∆94 form nucleosome core particles (NCPs). WT NCPs were made with 146 bp 601 DNA and histones using salt dialysis (SD NCP), direct addition microscale reconstitution (DNA + oct), and microscale reconstitution (DNA + H3/H4 + H2A/H2B) procedures. H4G94P and H4∆94 containing NCPs were formed using the microscale reconstitution (DNA + H3/H4 + H2A/H2B) procedure. Arrows indicate the positions of the WT and aberrant NCPs [37]. Lower panels are western blots made from the same gel with the indicated antibodies. (C) Electrophoretic analysis of NCP stability. NCPs formed by the microscale reconstitution (DNA + H3/H4 + H2A/H2B) procedure with WT histones, H4G94P, or H4∆94, were incubated with 200, 400, or 600 mM NaCl in the buffers for 1 h prior to electrophoresis. Arrows indicate the positions of the NCPs, H2A/H2B-DNA complexes, H3/H4-DNA complexes (tetrasomes), and free DNA.