TY - JOUR TI - An assay for de novo kinetochore assembly reveals a key role for the CENP-T pathway in budding yeast AU - Lang, Jackie AU - Barber, Adrienne AU - Biggins, Sue A2 - Musacchio, Andrea VL - 7 PY - 2018 DA - 2018/08/17 SP - e37819 C1 - eLife 2018;7:e37819 DO - 10.7554/eLife.37819 UR - https://doi.org/10.7554/eLife.37819 AB - Chromosome segregation depends on the kinetochore, the machine that establishes force-bearing attachments between DNA and spindle microtubules. Kinetochores are formed every cell cycle via a highly regulated process that requires coordinated assembly of multiple subcomplexes on specialized chromatin. To elucidate the underlying mechanisms, we developed an assay to assemble kinetochores de novo using centromeric DNA and budding yeast extracts. Assembly is enhanced by mitotic phosphorylation of the Dsn1 kinetochore protein and generates kinetochores capable of binding microtubules. We used this assay to investigate why kinetochores recruit the microtubule-binding Ndc80 complex via two receptors: the Mis12 complex and CENP-T. Although the CENP-T pathway is non-essential in yeast, we demonstrate that it becomes essential for viability and Ndc80c recruitment when the Mis12 pathway is crippled by defects in Dsn1 phosphorylation. Assembling kinetochores de novo in yeast extracts provides a powerful and genetically tractable method to elucidate critical regulatory events in the future. KW - kinetochore KW - budding yeast KW - assembly assay KW - microtubule KW - CENP-T JF - eLife SN - 2050-084X PB - eLife Sciences Publications, Ltd ER -