Direct observation of coordinated assembly of individual native centromeric nucleosomes

Eukaryotic chromosome segregation requires the kinetochore, a megadalton-sized machine that forms on specialized centromeric chromatin containing CENP‑A, a histone H3 variant. CENP‑A deposition requires a chaperone protein HJURP that targets it to the centromere, but it has remained unclear whether HJURP has additional functions beyond CENP‑A targeting and why high AT DNA content, which disfavors nucleosome assembly, is widely conserved at centromeres. To overcome the difficulties of studying nucleosome formation in vivo, we developed a microscopy assay that enables direct observation of de novo centromeric nucleosome recruitment and maintenance with single molecule resolution. Using this assay, we discover that CENP‑A can arrive at centromeres without its dedicated centromere-specific chaperone HJURP, but stable incorporation depends on HJURP and additional DNA-binding proteins of the inner kinetochore. We also show that homopolymer AT runs in the yeast centromeres are essential for efficient CENP‑A deposition. Together, our findings reveal requirements for stable nucleosome formation and provide a foundation for further studies of the assembly and dynamics of native kinetochore complexes.