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Vol. 18, Issue 8, 2805-2816, August 2007

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Spc24 and Stu2 Promote Spindle Integrity When DNA Replication Is Stalled

Lina Ma^*,, Jennifer McQueen^*,, Lara Cuschieri, Jackie Vogel^,, and Vivien Measday

*Genetics Graduate Program and Wine Research Centre/Michael Smith Laboratories, Faculty of Land and Food Systems, University of British Columbia, Vancouver, British Columbia, V6T 1Z4 Canada; and Department of Biology and Developmental Biology Research Initiative, McGill University, Montreal, QC, H3A 1B1 Canada

Submitted October 2, 2006; Revised May 8, 2007; Accepted May 9, 2007
Monitoring Editor: Tim Stearns

The kinetochore, a protein complex that links chromosomes to microtubules (MTs), is required to prevent spindle expansion during S phase in budding yeast, but the mechanism of how the kinetochore maintains integrity of the bipolar spindle before mitosis is not well understood. Here, we demonstrate that a mutation of Spc24, a component of the conserved Ndc80 kinetochore complex, causes lethality when cells are exposed to the DNA replication inhibitor hydroxyurea (HU) due to premature spindle expansion and segregation of incompletely replicated DNA. Overexpression of Stu1, a CLASP-related MT-associated protein or a truncated form of the XMAP215 orthologue Stu2 rescues spc24-9 HU lethality and prevents spindle expansion. Truncated Stu2 likely acts in a dominant-negative manner, because overexpression of full-length STU2 does not rescue spc24-9 HU lethality, and spindle expansion in spc24-9 HU-treated cells requires active Stu2. Stu1 and Stu2 localize to the kinetochore early in the cell cycle and Stu2 kinetochore localization depends on Spc24. We propose that mislocalization of Stu2 results in premature spindle expansion in S phase stalled spc24-9 mutants. Identifying factors that restrain spindle expansion upon inhibition of DNA replication is likely applicable to the mechanism by which spindle elongation is regulated during a normal cell cycle.

The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org).

Address correspondence to: Vivien Measday (vmeasday{at}interchange.ubc.ca).

Abbreviations used: CEN, centromere; ChIP, chromatin immunoprecipitation; CFP, cyan fluorescent protein; GFP, green fluorescent protein; HCS, high copy suppressor; HU, hydroxyurea; MT, microtubule; SPB, spindle pole body; Ts, temperature-sensitive; VFP, venus fluorescent protein.