The Cik1/Kar3 Motor Complex Is Required for the Proper Kinetochore–Microtubule Interaction After Stressful DNA Replication

Abstract

In budding yeast Saccharomyces cerevisiae, kinetochores are attached by microtubules during most of the cell cycle, but the duplication of centromeric DNA disassembles kinetochores, which results in a brief dissociation of chromosomes from microtubules. Kinetochore assembly is delayed in the presence of hydroxyurea, a DNA synthesis inhibitor, presumably due to the longer time required for centromeric DNA duplication. Some kinetochore mutants are sensitive to stressful DNA replication as these kinetochore proteins become essential for the establishment of the kinetochore-microtubule interaction after treatment with hydroxyurea. To identify more genes required for the efficient kinetochore-microtubule interaction under stressful DNA replication conditions, we carried out a genome-wide screen for yeast mutants sensitive to hydroxyurea. From this screen, cik1 and kar3 mutants were isolated. Kar3 is the minusend-directed motor protein; Cik1 binds to Kar3 and is required for its motor function. After exposure to hydroxyurea, cik1 and kar3 mutant cells exhibit normal DNA synthesis kinetics, but they display a significant anaphase entry delay. Our results indicate that cik1 cells exhibit a defect in the establishment of chromosome bipolar attachment in the presence of hydroxyurea. Since Kar3 has been shown to drive the poleward chromosome movement along microtubules, our data support the possibility that this chromosome movement promotes chromosome bipolar attachment after stressful DNA replication.

The current view for the silencing of the spindle assembly checkpoint

“…61 In a genomewide screen for yeast deletion mutants that are sensitive to stressful DNA replication, both cik1Δ and kar3Δ mutants were isolated, presumably due to the defect in chromosome bipolar attachment. 62 Our further analysis indicates that the loss of function of Cik1/Kar3 increases the chance of syntelic attachment, although the mechanism for this incorrect attachment remains to be determined. Moreover, we found that overexpression of the coiled-coil domain of Cik1 (Cik1-CC) disrupts Cik1-Kar3 interaction, which allows us to conditionally inactivate the Cik1/ Kar3 motor complex and induce syntelic attachments.…”

The signaling network that silences the spindle assembly checkpoint upon the establishment of chromosome bipolar attachment

“…Moreover, overexpression of the coiled-coil domain of CIK1 (CIK1-CC) disrupts Cik1-Kar3 interaction and induces an anaphase entry delay that depends on both Ipl1 and Sgo1 (9,18). Therefore, we also compared the Mad1 phosphorylation kinetics in wild-type (WT), ipl1, and sgo1 mutant cells overexpressing CIK1-CC.…”

Slk19 clusters kinetochores and facilitates chromosome bipolar attachment

“…To reestablish the KT-MT interaction, the disconnected kinetochores are first captured by a microtubule and then moved to the vicinity of one spindle pole through Cik1/Kar3-mediated transport (Tanaka et al. , 2005, 2007), which may facilitate chromosome bipolar attachment (Liu et al. , 2011).…”