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bioRxiv · 10.1101/2023.01.20.524981

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

Popchock, A.·Larson, J.·Dubrulle, J.·Asbury, C. L.·Biggins, S.
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Abstract

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.

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Popchock, A., Larson, J., Dubrulle, J., Asbury, C. L., Biggins, S.. 2023-01-21. Single molecule visualization of native centromeric nucleosome formation reveals coordinated deposition by kinetochore proteins and centromere DNA sequence. https://doi.org/10.1101/2023.01.20.524981

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