Abstract
Genomic DNA in eukaryotes is organized into chromatin through association with core histones to form nucleosomes, each distinguished by their DNA sequences and histone variants. Here, we used a single-chain antibody fragment (scFv) derived from the anti-nucleosome antibody mAb PL2-6 to stabilize human CENP-A nucleosome containing a native α-satellite DNA and solved its structure by the cryo-electron microscopy (cryo-EM) to 2.6 Å resolution. In comparison, the corresponding cryo-EM structure of the free CENP-A nucleosome could only reach 3.4 Å resolution. We find that scFv binds to a conserved acidic patch on the histone H2A-H2B dimer without perturbing the nucleosome structure. Our results provide an atomic resolution cryo-EM structure of a nucleosome and insight into the structure and function of the CENP-A nucleosome. The scFv approach is applicable to the structural determination of other native-like nucleosomes with distinct DNA sequences.
Highlights
Genomic DNA in eukaryotes is organized into chromatin through association with core histones to form nucleosomes, each distinguished by their DNA sequences and histone variants
Gel shift assay and isothermal titration calorimetric experiments showed that scFv bound to the Nucleosome core particles (NCP) containing Drosophila core histones and 147 bp Widom “601” (W601) DNA, termed NCPH3, W601, with 2:1 stoichiometry and a dissociation constant (Kd) of ~190 nM for each binding site (Supplementary Fig. 1b, c)
The outward shifts of the DNA at the entry and exit sites in the CENP-A NCP would lead to a more open conformation of the linker DNA in the nucleosome in comparison with those in the H3 nucleosome bound to a linker histone (Fig. 5a)[26,27]
Summary
Genomic DNA in eukaryotes is organized into chromatin through association with core histones to form nucleosomes, each distinguished by their DNA sequences and histone variants. Single particle cryo-EM studies of NCPCENP-A,NAS free nucleosomes or those in complex with other proteins have mainly relied on the Widom “601” (W601) DNA, which was scFv selected in vitro for high affinity binding to the core histones[5]. We overcome this hurdle by using a single-chain antibody fragment (scFv) to stabilize the nucleosome. We determine the cryo-EM structure of the human centromeric nucleosome containing CENP-A and a native α-satellite DNA sequence at 2.6 Å resolution. The results and the scFv method present here pave an avenue for the structural determination of nucleosomes with natural DNA sequences at atomic resolution by cryo-EM
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