Student Theses and Dissertations

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RU Laboratory

Cross Fred Laboratory


Cyclin-dependent kinase (Cdk), Clb2kd, Saccharomyces cerevisiae, mitosis, cell cycle ordering


In eukaryotes, DNA replication, mitosis, and cytokinesis are all regulated by Cyclin-dependent kinase (Cdk). Cyclin/Cdk complexes promote replication origin firing and mitotic entry, and conversely, inhibit pre-replication origin loading and exit from mitosis. Cyclin synthesis and degradation, Cdk phosphorylation and Cdk inhibitors are controlled such that Cdk activity oscillates once per cell cycle. Little is known about the quantitative relationship between the level of Cdk activity and the occurrence, rate, and coordination of cell cycle events. We have addressed this question in Saccharomyces cerevisiae by introducing titrated levels of undegradable mitotic B-cyclin (Clb2kd) in cells prior to release of a metaphase block. We calibrated single-cell Clb2kd levels to the peak level of endogenous Clb2 attained in a normal cell cycle and examined the ability of differing amounts of Clb2kd to affect completion of mitosis and initiation of the next cell cycle. Clb2kd delays mitotic exit, and interferes with α-factor response and bipolar spindle assembly in the subsequent cell cycle. Distinct cell cycle events have different B-cyclin inhibitory thresholds; significantly, an amount of Clb2kd equal to the peak endogenous Clb2 level attained in a normal cell cycle only marginally restrains any aspect delays most aspects of mitotic exit. Genetic experiments and in vitro kinase assays suggest that posttranslational regulation of Cdk activity has only modest effects on these results; thus, Clb2kd levels are reliable indicators of Clb2kd/Cdk activity. The ability of Cdk to both activate and inhibit sequential processes, combined with the oscillation of cyclin/Cdk activity, suggested ‘ratchet’ models to explain cell cycle ordering. If the Cdk activating threshold for the first event is greater than the inhibitory threshold for the second, then these thresholds form an activity ratchet that couples the sequence to Cdk oscillation. These models account for considerable data, but they have generally been tested by contrasting absence of Cdk activity to gross overexpression. We found that the normal peak level of Clb2 promotes efficient mitotic entry but cannot stably restrain mitotic exit or subsequent DNA replication. These results challenge ratchet models, and suggest that Cdk activity oscillation is insufficient to explain ordering of cell cycle events.


A thesis presented to the faculty of The Rockefeller University in partial fulfillment of the requirements for the degree of Doctor of Philosophy.

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