An assay for de novo kinetochore assembly reveals a key role for the CENP-T pathway in budding yeast

Abstract

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.

The COMA complex is required for positioning Ipl1 activity proximal to Cse4 nucleosomes in budding yeast

“…The Ame1/Okp1 heterodimer in complex with the Cse4-NCP retained the ability to associate with the outer kinetochore MTW1c ( Figure 2C) (Hornung et al, 2014). As MTW1c recruitment by Mif2 and Cnn1 are redundant pathways and become only essential if one of both is compromised (Hornung et al, 2014, Lang et al, 2018, Ame1/Okp1 is the sole essential link of the centromeric nucleosomes to the outer kinetochore which emphasizes its role as cornerstone of kinetochore assembly at the budding yeast point centromere.…”

“…In contrast, Saccharomyces cerevisiae possesses point centromeres which are characterized by a specific ~125 bp DNA sequence wrapped around a single Cse4 containing histone octamer. The kinetochore unit built on this point centromere stays assembled and bound to a single microtubule throughout almost the whole cell cycle (Pluta et al, 1995, Biggins, 2013, Lang et al, 2018.…”

The structure of the yeast Ctf3 complex

“…Both Scc2/4 recruitment and stable microtubule binding at later stages of the cell cycle depend on the conserved Ctf3/CENP-I complex (Ctf3c) (Hara et al, 2018;Lang et al, 2018;Natsume et al, 2013). The Ctf3c namesake, Ctf3, is a 733 amino-acid residue protein predicted to consist almost entirely of HEAT repeats (Huntingin, elongation factor 3 (EF3), protein phosphatase 2A (PP2A), and yeast kinase TOR1 repeats) (Basilico et al, 2014).…”

A dCas9/CRISPR-based targeting system identifies a central role for Ctf19 in kinetochore-derived suppression of meiotic recombination

“…This biochemical characteristic can lead to pleiotropic loss of several kinetochore subunits 93 upon experimental interference with single components. For example, many Ctf19c subunits are 94 co-dependent for their localization to the centromere (Lang et al, 2018, Pekgoz Altunkaya et al, 95 2016, Pot et al, 2003. This behaviour has complicated delineating individual contributions of 96 single kinetochore components to specific functional pathways, including the regulation of local 97 crossover suppression.…”