Single molecule visualization of native centromeric nucleosome formation reveals coordinated deposition by kinetochore proteins and centromere DNA sequence.

Eukaryotic chromosome segregation requires the kinetochore, a conserved megadalton-sized machine that forms on specialized centromeric chromatin containing CENP-A, a histone H3 variant. CENP-A deposition requires a conserved 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 at single molecule resolution. Using this assay, we discover that CENP-A can arrive at centromeres without its chaperone, but stable incorporation depends on HJURP and on 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.