(A) Flow diagram for preparation of the CD fraction. (B) Chromatin assembly using the CD fraction: outline of experiment. Plasmid DNA was relaxed with topoisomerase I (Top I, + and ++) prior to assembly or not (−). RT, room temperature; MNase, micrococcal nuclease. (C) Assembly with the CD fraction results in supercoiling of previously relaxed templates. After assembly, plasmid DNA was reisolated, resolved by 1% agarose gel electrophoresis, and stained with ethidium bromide. SC, supercoiled plasmid; R/OC, relaxed/open circular plasmid. Lanes 1 to 3 show the input DNA for the assembly reactions in lanes 4 to 6. (D) Assembly of physiologically spaced nucleosomal arrays does not require exogenous topoisomerase. The reaction products were treated with 0.37, 1.1, or 3.3 U of micrococcal nuclease/ml, resolved by agarose gel electrophoresis, and visualized with ethidium bromide. M, 1 kbp plus DNA ladder (Stratagene). (E) RE protection assay: outline of experiment. (F) DNA assembled into chromatin by using the CD fraction is protected from RE digestion. The diagram shows the relative positions of cutting sites for enzymes used. Products from reactions with naked (N) or assembled (A) DNA were resolved by agarose gel electrophoresis and visualized with ethidium bromide. The 3.3-kbp linear plasmid is shown in lanes 1 and 2. M, 1 kbp plus DNA ladder (Stratagene).

Chromatin assembly requires exogenous histones. (A) Outline of experiment. Plasmid DNA was incubated with only CD fraction or with CD fraction and yeast (y) or Drosophila (D) core histones. RT, room temperature; MNase, micrococcal nuclease. (B) Plasmid supercoiling. After assembly, plasmid DNA was reisolated, resolved by 1% agarose gel electrophoresis and stained with ethidium bromide. SC, supercoiled plasmid; R/OC, relaxed/open circular plasmid. (C) Micrococcal nuclease digestion assay. The product of the micrococcal nuclease digestions were resolved by agarose gel electrophoresis and visualized with ethidium bromide. M, 1 kbp plus DNA ladder.

Assembly of nucleosome arrays is protein dependent and requires ATP. (A) Assembly of nucleosome arrays is protein dependent. Reactions were performed as summarized in the flow diagram. The CD fraction was pretreated for 15 min at 30°C by itself (lanes 1 and 2), with proteinase K (PK, 0.3 μg/μl; lanes 3 and 4), or with RNase A (0.12 μg/μl; lanes 7 and 8) or was incubated at 55°C for 15 min (lanes 5 and 6). The proteinase inhibitor AEBSF (15 mM) was added to the proteinase K-digested sample after pretreatment. The pretreated samples were then mixed with core histones for assembly. Micrococcal digestion assays were performed and analyzed by agarose gel electrophoresis, and the DNA was visualized with ethidium bromide. RT, room temperature; MNase, micrococcal nuclease; M, 1 kbp plus DNA ladder. (B) Assembly of nucleosome arrays is ATP dependent. Assembly reactions were performed according to the flow diagram, in the absence (−) or presence (+) of ATP, creatine phosphate, and creatine kinase. All reactions contained 6 mM MgCl₂. The ATP regeneration system and the CD fraction were pretreated with apyrase (start, lanes 7 to 9), or apyrase was added to the assembly reaction for 45 min after 1 h of assembly (after, lanes 10 to 12). Micrococcal digestions were performed, and the products were resolved by agarose gel electrophoresis and visualized with ethidium bromide. RT, room temperature; MNase, micrococcal nuclease; M, 1 kbp plus DNA ladder.

Yeast core histones form extensive nucleosomal arrays when ATP-dependent nucleosomal mobilization is inhibited. (A) Coomassie blue staining of yeast histones resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The migration of molecular mass markers is indicated on the left in kilodaltons. (B) Flow diagram of reactions, which were performed by using the standard reaction cocktail. After 75 min, apyrase was added to half of the samples (lanes 3 and 4 in panel C), and all of the reactions were incubated for an additional 45 min. RT, room temperature; MNase, micrococcal nuclease. (C) Assembly products were assayed by micrococcal nuclease digestion, followed by agarose gel electrophoresis. The DNA was visualized with ethidium bromide. M, 1 kbp plus DNA ladder.

Screening of CD fraction from 10 deletion mutants for chromatin assembly activity (plasmid supercoiling). Chromatin templates were assembled with CD fractions prepared from wild-type cells (WT; BY4741) or 10 isogenic strains harboring deletions of the indicated genes. After assembly reactions, the plasmid DNA was reisolated, resolved by agarose gel electrophoresis, and stained with ethidium bromide. In Fig. 6 and 7, reactions were performed with the final concentration of extract at 1.2 mg/ml. The activities of mutant and wild-type (lane 2) samples were compared. Relaxed plasmid DNA was run in lane 1. SC, supercoiled plasmid; R/OC, relaxed/open circular plasmid.

Screen of capacity to assemble nucleosome arrays. Two wild-type (WT; BY4741) CD fractions are shown (lanes 1 to 3 and 19 to 21). Array assembly was monitored by partial micrococcal nuclease digestion (MNase) followed by agarose gel electrophoresis and staining with ethidium bromide. M, 1 kbp plus DNA ladder.

Chromatin assembly in the CD fraction from asf1Δ cells. (A) Supercoiling assay of reactions with increasing amounts of CD fraction from wild-type (WT; lanes 2, 4, and 6) and asf1Δ (aΔ; lanes 3, 5, and 7) strains. Reactions were done with CD fraction at final concentrations of 0.7 (+), 1.0 (++), and 1.4 (+++) mg/ml. After assembly, the plasmid DNA was reisolated, resolved by agarose gel electrophoresis, and stained with ethidium bromide. To ensure that the supercoiling defect was not due to a lack of topoisomerase activity, the plasmid DNA was relaxed prior to assembly, and the reactions were supplemented with topoisomerase I. The relaxed input DNA was run in lane 1. SC, supercoiled plasmid; R/OC, relaxed/open circular plasmid. (B) Micrococcal nuclease digestion assay of chromatin assembled with CD fraction from wild-type (WT; lanes 1 to 3 and lanes 7 to 9) and asf1Δ (lanes 4 to 6 and lanes 10 to 12) strains, with the amount of CD fraction varied as in panel A (++ and +++). After micrococcal digestion, the DNA was reisolated, resolved by agarose gel electrophoresis, and stained with ethidium bromide. MNase, micrococcal nuclease; M, 1 kbp plus DNA ladder.

Chromatin assembly in the CD fraction from chd1Δ cells. (A) Supercoiling assay of reactions with increasing amounts of CD fraction from wild-type (WT; lanes 2, 4, and 6) and chd1Δ (cΔ; lanes 3, 5, and 7) strains. Reactions were done with CD fraction at final concentrations of 0.7 (+), 1.0 (++), and 1.4 (+++) mg/ml. After assembly the plasmid DNA was reisolated, resolved by agarose gel electrophoresis, and stained with ethidium bromide. The relaxed input DNA was run in lane 1. SC, supercoiled plasmid; R/OC, relaxed/open circular plasmid. (B) Micrococcal nuclease digestion assay with chromatin assembled with CD fraction from wild-type (WT; lanes 1 to 3 and lanes 7 to 9) and chd1Δ (lanes 4 to 6 and lanes 10 to 12) strains, with the amount of CD fraction varied as in panel A (++ and +++). After micrococcal digestion, the DNA was reisolated and resolved by agarose gel electrophoresis. The DNA was visualized with ethidium bromide. MNase, micrococcal nuclease; M, 1 kbp plus DNA ladder.