Abstract
Premature anaphase onset is prevented by the mitotic checkpoint through production of a "wait anaphase" inhibitor(s) that blocks recognition of cyclin B and securin by Cdc20-activated APC/C, an E3 ubiquitin ligase that targets them for destruction. Using physiologically relevant levels of Mad2, Bub3, BubR1, and Cdc20, we demonstrate that unattached kinetochores on purified chromosomes catalytically generate a diffusible Cdc20 inhibitor or inhibit Cdc20 already bound to APC/C. Furthermore, the chromosome-produced inhibitor requires both recruitment of Mad2 by Mad1 that is stably bound at unattached kinetochores and dimerization-competent Mad2. We show that purified chromosomes promote BubR1 binding to APC/C-Cdc20 by acting directly on Mad2, but not BubR1. Our results support a model in which immobilized Mad1/Mad2 at kinetochores provides a template for initial assembly of Mad2 bound to Cdc20 that is then converted to a final mitotic checkpoint inhibitor with Cdc20 bound to BubR1.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, Non-U.S. Gov't
MeSH terms
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Anaphase / physiology*
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Anaphase-Promoting Complex-Cyclosome
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Calcium-Binding Proteins / chemistry
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Calcium-Binding Proteins / genetics
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Calcium-Binding Proteins / metabolism*
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Cdc20 Proteins
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Cell Cycle Proteins / chemistry
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Cell Cycle Proteins / genetics
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Cell Cycle Proteins / metabolism*
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Chromosomes / metabolism
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Dimerization
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HeLa Cells
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Humans
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Kinetochores / metabolism*
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Mad2 Proteins
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Nuclear Proteins / genetics
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Nuclear Proteins / metabolism
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Poly-ADP-Ribose Binding Proteins
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Protein Serine-Threonine Kinases / genetics
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Protein Serine-Threonine Kinases / metabolism*
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Recombinant Proteins / genetics
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Recombinant Proteins / metabolism
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Repressor Proteins / chemistry
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Repressor Proteins / genetics
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Repressor Proteins / metabolism*
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Ubiquitin-Protein Ligase Complexes / antagonists & inhibitors*
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Ubiquitin-Protein Ligase Complexes / metabolism
Substances
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BUB3 protein, human
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Calcium-Binding Proteins
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Cdc20 Proteins
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Cell Cycle Proteins
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MAD1L1 protein, human
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MAD2L1 protein, human
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Mad2 Proteins
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Nuclear Proteins
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Poly-ADP-Ribose Binding Proteins
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Recombinant Proteins
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Repressor Proteins
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CDC20 protein, human
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Ubiquitin-Protein Ligase Complexes
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Anaphase-Promoting Complex-Cyclosome
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BUB1 protein, human
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Bub1 spindle checkpoint protein
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Protein Serine-Threonine Kinases