Functional analysis of the spindle-checkpoint proteins using an in vitro ubiquitination assay

Methods in Molecular Biology
Zhanyun Tang, Hongtao Yu

Abstract

The spindle checkpoint helps to ensure the fidelity of chromosome segregation during mitosis and meiosis. In response to sister chromatids not properly attached to the mitotic spindle, this checkpoint blocks the activity of a large ubiquitin protein ligase complex, called the anaphase-promoting complex (APC) or cyclosome. This chapter describes the detailed protocols of an in vitro ubiquitination assay reconstituted with purified APC, cofactors, other ubiquitination enzymes, and the spindle-checkpoint proteins. This assay is extremely useful in dissecting the biochemical functions of various spindle-checkpoint proteins.

Citations

Dec 30, 2004·Proceedings of the National Academy of Sciences of the United States of America·Baoying LiuChou-Zen Giam
Dec 21, 2005·Proceedings of the National Academy of Sciences of the United States of America·Engin OzkanJohann Deisenhofer
Oct 24, 2012·Proceedings of the National Academy of Sciences of the United States of America·Wei TianXuelian Luo
Nov 19, 2008·The Journal of Biological Chemistry·Zemfira KaramyshevaHongtao Yu
Oct 1, 2005·Genes & Development·Marito ArakiMaki Asano
Dec 24, 2011·Journal of Cell Science·Pablo Lara-GonzalezStephen S Taylor
Jul 23, 2008·Molecular Cancer Therapeutics·Sigrid A RajasekaranAyyappan K Rajasekaran

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Cell cycle checkpoints are a series of complex checkpoint mechanisms that detect DNA abnormalities and ensure that DNA replication and repair are complete before cell division. They are primarily regulated by cyclins, cyclin-dependent kinases, and the anaphase-promoting complex/cyclosome. Here is the latest research.

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