Characterization of CDC16-183: A novel APC mutant which activates the spindle checkpoint [electronic resource].

Lai, Lisa Ann L.
180 p.
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Mode of access: World Wide Web.
Genomic stability is ensured in part by the spindle damage checkpoint, which arrests the cell cycle in response to defects in spindle structure or chromosome attachments. Activation of the spindle checkpoint prevents premature sister chromatid separation by inhibition of the anaphase promoting complex or cyclosome (APC). Through its role as an E3 ubiquitin ligase the APC facilitates anaphase onset and mitotic progression by the ubiquitin-mediated degradation of various substrate proteins. We employed a mutagenesis strategy to identify novel APC mutants which were defective in spindle checkpoint regulation in the budding yeast, Saccharomyces cerevisiae. We focused on one subunit, CDC16, due to recent evidence demonstrating that phosphorylation of Cdcl6p correlates with APC activation. Furthermore, CDC16 has been implicated in response to the DNA damage and re-replication checkpoints. We have identified a novel allele, cdc16-183, which displayed temperature sensitivity and supersensitivity to nocodazole, an anti-microtubule agents which activates the spindle checkpoint. Unlike spindle checkpoint mutants, however, cdc16-183 arrested normally at metaphase in spindle damaging conditions. We have shown that cdc16-183 is supersensitive to nocodazole as a consequence of its inability to overcome, or adapt to, a checkpoint-induced metaphase arrest. Furthermore, both the temperature conditionality and nocodazole supersensitivity of cdc16-183 can be relieved by deletion of the spindle checkpoint gene, MAD2. The metaphase arrest of cdc16-183 at the restrictive temperature is suppressed in cdc16-183 mad2Delta double mutants, suggesting that this mutant may activate the spindle checkpoint in the absence of exogenous damaging agents. We have verified that G1 APCcdc16-183 functions normally, confirming that these defects cannot be explained by impaired proteolytic activity. This work suggests a novel role for the APC in spindle checkpoint activation and a requirement for APC function in the establishment of spindle and/or chromosome structure in both normal and perturbed cell cycles.
Source: Dissertation Abstracts International, Volume: 62-11, Section: B, page: 4917.
Adviser: Sandra L. Holloway.
Thesis (Ph.D.)--University of Pennsylvania, 2001.
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School code: 0175.
University of Pennsylvania.
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Dissertation Abstracts International 62-11B.
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Restricted for use by site license.
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