![Figure 6. CAL1 is a CENP-A–specific nucleosome assembly factor. (A) CENP-A101–225–H4 was assembled on circular relaxed plasmid (pCR2.1-4 [CEN3 + CEN6]) using increasing amounts of CAL11–96, CAL11–132, or CAL11–160 in the presence of topoisomerase I. The extracted DNA samples were analyzed on agarose gel and stained with SYBR gold. Lane 1: supercoiled plasmid; lane 2: relaxed plasmid; lane 3: CENP-A101–225–H4, H2A–H2B but no CAL1; lanes 4–7: relaxed plasmid and CENP-A101–225–H4, H2A–H2B with CAL11–96; lanes 8–11: CENP-A101–225–H4, H2A–H2B with CAL11–132; lanes 12–15: CENP-A101–225–H4, H2A–H2B with CAL11–160. (B) CENP-A–containing nucleosomes and histone H3 nucleosomes were assembled using histone proteins, CAL11–160, or yeast Nap1 and pGEM3Z-601 plasmid. Lane 1: supercoiled plasmid; lane 2: relaxed plasmid; lanes 3, 6, and 9: mock reaction without CAL11–160 or Nap1; lanes 4 and 5: H3–H4, H2A–H2B with CAL11–160; lanes 7 and 8: CENP-A101–225–H4, H2A-H2B with CAL11–160; lanes 10 and 11: H3–H4, H2A–H2B with Nap1; lanes 12 and 13: CENP-A101–225–H4, H2A–H2B with Nap1. (C) CAL1-assembled CENP-A nucleosomes are negatively supercoiled. CENP-A nucleosomes (lane CENP-A) were assembled by CAL11–160 in the presence of topoisomerase I and H2A–H2B dimers, whereas histone H3 nucleosomes (lane H3) were assembled by Nap1. Controls are: supercoiled plasmid (superc.), relaxed plasmid (relaxed), and relaxed plasmid without histones or Nap1, but treated and processed as in the H3 and CENP-A nucleosome assembly reactions (mock). The samples were analyzed on agarose gels with or without 1 µg/ml chloroquine. Boxes highlight the decrease in migration that occurs in the presence of chloroquine in both H3 and CENP-A nucleosomes. (A–C) S, supercoiled; R, relaxed plasmid. (D) Diagram depicting the expected migration patterns for negatively and positively supercoiled DNA separated by 2D gel electrophoresis (Tachiwana et al., 2011). Left gel: H3 nucleosomes assembled with Nap1; right gel: CENP-A nucleosomes assembled with CAL11–160. (E) Mononucleosomes were assembled on 147-bp Widom DNA with Nap1 or CAL11–160 and digested with MNase for 30 or 120 s. Native PAGE of samples shown. Lanes 1–3: DNA only; lanes 4 and 5: H3 nucleosomes assembled by Nap1; lanes 6 and 7: CENP-A nucleosomes assembled by Nap1; lanes 8 and 9: CENP-A nucleosomes assembled by CAL11–160.](https://cdn.rupress.org/rup/content_public/journal/jcb/204/3/10.1083_jcb.201305036/6/jcb_201305036r_fig6.jpeg?Expires=1771605572&Signature=NAitepRhebvo6uqfEtwnEeX6rjY9tdqCpmQ83Pca1dqM3sMA4vH5~jIR1LoFDpUzoEnsFg8jgAVVsJFJYfrJjvo6efu61AxSqS1NOJyOSDd7bBAjHZB-rTBnqEtdFn4EeygxVwqu-ufaUuuuHNSWYgU63N3N37dZ1IPJAGkG5dpAl7CsTJxb0mIt7GFPXYw~whOnL3mAlmLi91Td0Ne8QQJwSVnLqa-DPgPpWUD5DqFSeLD~mIisQNwhYYvDOFrLkHlDAbgsDzZrQxTcLwihH5mdOszwN7qBe1VpwNVSs~jLeVTPoNFnsE5Nf6whvbrkBcG6SahI6GupwIHETxvcVw__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
CAL1 is a CENP-A–specific nucleosome assembly factor. (A) CENP-A101–225–H4 was assembled on circular relaxed plasmid (pCR2.1-4 [CEN3 + CEN6]) using increasing amounts of CAL11–96, CAL11–132, or CAL11–160 in the presence of topoisomerase I. The extracted DNA samples were analyzed on agarose gel and stained with SYBR gold. Lane 1: supercoiled plasmid; lane 2: relaxed plasmid; lane 3: CENP-A101–225–H4, H2A–H2B but no CAL1; lanes 4–7: relaxed plasmid and CENP-A101–225–H4, H2A–H2B with CAL11–96; lanes 8–11: CENP-A101–225–H4, H2A–H2B with CAL11–132; lanes 12–15: CENP-A101–225–H4, H2A–H2B with CAL11–160. (B) CENP-A–containing nucleosomes and histone H3 nucleosomes were assembled using histone proteins, CAL11–160, or yeast Nap1 and pGEM3Z-601 plasmid. Lane 1: supercoiled plasmid; lane 2: relaxed plasmid; lanes 3, 6, and 9: mock reaction without CAL11–160 or Nap1; lanes 4 and 5: H3–H4, H2A–H2B with CAL11–160; lanes 7 and 8: CENP-A101–225–H4, H2A-H2B with CAL11–160; lanes 10 and 11: H3–H4, H2A–H2B with Nap1; lanes 12 and 13: CENP-A101–225–H4, H2A–H2B with Nap1. (C) CAL1-assembled CENP-A nucleosomes are negatively supercoiled. CENP-A nucleosomes (lane CENP-A) were assembled by CAL11–160 in the presence of topoisomerase I and H2A–H2B dimers, whereas histone H3 nucleosomes (lane H3) were assembled by Nap1. Controls are: supercoiled plasmid (superc.), relaxed plasmid (relaxed), and relaxed plasmid without histones or Nap1, but treated and processed as in the H3 and CENP-A nucleosome assembly reactions (mock). The samples were analyzed on agarose gels with or without 1 µg/ml chloroquine. Boxes highlight the decrease in migration that occurs in the presence of chloroquine in both H3 and CENP-A nucleosomes. (A–C) S, supercoiled; R, relaxed plasmid. (D) Diagram depicting the expected migration patterns for negatively and positively supercoiled DNA separated by 2D gel electrophoresis (Tachiwana et al., 2011). Left gel: H3 nucleosomes assembled with Nap1; right gel: CENP-A nucleosomes assembled with CAL11–160. (E) Mononucleosomes were assembled on 147-bp Widom DNA with Nap1 or CAL11–160 and digested with MNase for 30 or 120 s. Native PAGE of samples shown. Lanes 1–3: DNA only; lanes 4 and 5: H3 nucleosomes assembled by Nap1; lanes 6 and 7: CENP-A nucleosomes assembled by Nap1; lanes 8 and 9: CENP-A nucleosomes assembled by CAL11–160.
