Figure 3.

Mono-prenucleosomes contain all four core histones and are distinct from hexasomes. (A) Sucrose gradient sedimentation analysis reveals that mono-prenucleosomes contain all four core histones. Mono-prenucleosomes were prepared by either NAP1-mediated deposition (left panels) or salt dialysis (right panels) and then subjected to 10%–30% (w/v) (left to right) sucrose gradient sedimentation in a Beckman SW41 rotor (32,000 rpm for 18 h at 4°C). The arrows indicate the direction of sedimentation. (Top panels) The presence of mono-prenucleosomes was detected by native polyacrylamide gel electrophoresis and ethidium bromide staining of the DNA. (Bottom panels) The protein composition was analyzed by SDS–polyacrylamide gel electrophoresis and silver staining. The top two fractions and the bottom fraction did not contain histones (for example, see Supplemental Fig. S2A) and are not included. The sedimentation of the free core histones relative to prenucleosomal histones is shown in Supplemental Figure S2A. (B) Mono-prenucleosomes contain two copies of H2A and thus appear to contain a core histone octamer rather than hexamer. Mono-prenucleosomes were reconstituted with recombinant core histones onto the 80-bp genomic DNA by NAP1-mediated histone deposition. The H2A species were a combination of wild-type H2A and Strep-H2A at a 3:1 ratio of H2A:Strep-H2A. Prenucleosomes containing Strep-H2A were pulled down with streptavidin beads and then analyzed by Western blot with antibodies against histone H2A. An H2A Western blot and silver-stained SDS gel are also shown for the input samples. The Western blots were detected and quantitated by using ³²P-labeled protein A.