Fig. 3

Three globular domains of HP1BP3 bind redundantly to NCPs. (A) Preparation of the globular domain mutants of HP1BP3. HP1BP3 and its mutants lacking two of three globular domains, G1 (amino acids 152–227), G2 (amino acids 254–336), and G3 (amino acids 341–409), are represented schematically. ΔG12, ΔG13, and ΔG23 lack two globular domains as indicated. HP1BP3 mutant lacking all globular domains, ΔG123, was also prepared. The Flag-tagged HP1BP3 proteins were expressed in 293T cells and purified with Flag-M2 agarose beads. The proteins (500 ng) were separated by 10% SDS-PAGE and visualized by CBB staining. Lanes M indicate molecular weight markers. (B) NCP binding activity of HP1BP3 globular domain mutants. NCPs (0.1 pmole) were incubated with increasing amounts wild type HP1BP3 (lanes 2–4), HP1BP3ΔG12 (lanes 5–7), HP1BP3ΔG13 (lanes 8–10), and HP1BP3ΔG23 (lanes 11–13) (0.04, 0.1, and 0.2 pmole for each protein) and separated by 6% native PAGE in 0.5×TBE. DNA was visualized by Gel Red staining. The bands for NCPs and NCP-HP1BP3 complexes were quantitatively analyzed by Image J and the data are shown at the right side of the panel. (C) MNase digestion assay of NCP-HP1BP3 mutant complexes. NCPs (0.2 pmole) incubated without (buffer, lanes 1 and 12) or with Flag-HP1BP3 ΔG12, ΔG13, and ΔG23 (lanes 2–6, 7–11, and 13–17, respectively) (0.05, 0.1, 0.2, 0.3 and 0.4 pmole) were treated with micrococcus nuclease (MNase, 1 unit) at 37 °C for 1 min (right panel), followed by gel extraction and electrophoresis on PAGE. Positions of molecular weight markers are indicated at the right side of the panels. In lane UC, DNA was extracted without MNase digestion. (D) The globular domains of HP1BP3 are essential for its NCP binding. NCPs (0.1 pmole) was incubated without (lane 1) or with increasing amounts of HP1BP3 wild type and HP1BP3 ΔG123 (lanes 2–5 and 6–9, respectively) (0.1, 0.2, 0.3, and 0.4 pmole) and the complexes were separated by native PAGE. Positions of NCPs, NCP-HP1BP3, and free DNA are shown at the right side of the panel. (E) HP1BP3 recognizes single NCP. NCPs were assembled on 196 bp–5 S rDNA and 250 bp-Widom DNA. The 196 bp-NCPs (lanes 1–4) and 250 bp-NCP (lanes 5–8) (0.1 pmole each) were mixed with increasing amount of HP1BP3 (0.2, 0.4, and 0.8 pmole). In lanes 9–12, 196 bp-NCP and 250 bp-NCP (0.1 pmole each) were mixed with increasing amounts of HP1BP3 (0.2, 0.4, and 0.8 pmole). The complexes were incubated and separated by 6% PAGE. DNA was visualized by Gel Red staining. Positions of 196 bp-NCPs and 250 bp-NCP are indicated at the left side of lane 1 and right side of lane 5, respectively, with filled arrowheads. Positions of 196 bp-NCP-HP1BP3 and 250 bp-NCP-HP1BP3 complexes are indicated at the left side of lane 2 and right side of lane 7, respectively, with blank arrowheads