ABSTRACT
Cohesion between sister chromatids occurs along the length of chromosomes, where it plays essential roles in chromosome segregation. We show here that the centromere, a cis-acting cohesion factor, directs the binding of Mcd1p, a cohesin subunit, to at least 2 kb regions flanking centromeres in a sequence-independent manner. The centromere is essential for the maintenance as well as the establishment of this cohesin domain. The efficiency of Mcd1p binding within the cohesin domain is independent of the primary nucleotide sequence of the centromere-flanking DNA but correlates with high A + T DNA content. Thus, the function of centromeres in the cohesion of centromere-proximal regions may be analogous to that of enhancers, nucleating cohesin complex binding over an extended chromosomal domain of A + T-rich DNA.
Subject(s)
Cell Cycle Proteins/metabolism , Centromere/metabolism , Chromatids/metabolism , DNA-Binding Proteins , Nuclear Proteins/metabolism , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/genetics , AT Rich Sequence , Binding Sites , Chromatin/isolation & purification , Chromosomal Proteins, Non-Histone , Chromosomes, Fungal/metabolism , DNA Nucleotidyltransferases/metabolism , Fungal Proteins/metabolism , Phosphoproteins , Polymerase Chain Reaction , Precipitin Tests , Protein Binding , Recombination, Genetic , CohesinsABSTRACT
The spindle checkpoint arrests cells in mitosis in response to defects in the assembly of the mitotic spindle or errors in chromosome alignment. We determined which spindle defects the checkpoint can detect by examining the interaction of mutations that compromise the checkpoint (mad1, mad2, and mad3) with those that damage various structural components of the spindle. Defects in microtubule polymerization, spindle pole body duplication, microtubule motors, and kinetochore components all activate the MAD-dependent checkpoint. In contrast, the cell cycle arrest caused by mutations that induce DNA damage (cdc13), inactivate the cyclin proteolysis machinery (cdc16 and cdc23), or arrest cells in anaphase (cdc15) is independent of the spindle checkpoint.
Subject(s)
Repressor Proteins , Saccharomyces cerevisiae/genetics , Spindle Apparatus/genetics , Cell Division/genetics , Cyclins/metabolism , DNA-Binding Proteins/genetics , Gene Expression Regulation, Fungal , Genes, Fungal/genetics , Genes, cdc/genetics , Hydrolysis , Kinetochores , Microtubules/genetics , Molecular Motor Proteins/genetics , Mutation , Saccharomyces cerevisiae/cytology , Tubulin/geneticsABSTRACT
The spindle checkpoint regulates the cell division cycle by keeping cells with defective spindles from leaving mitosis. In the two-hybrid system, three proteins that are components of the checkpoint, Mad1, Mad2, and Mad3, were shown to interact with Cdc20, a protein required for exit from mitosis. Mad2 and Mad3 coprecipitated with Cdc20 at all stages of the cell cycle. The binding of Mad2 depended on Mad1 and that of Mad3 on Mad1 and Mad2. Overexpression of Cdc20 allowed cells with a depolymerized spindle or damaged DNA to leave mitosis but did not overcome the arrest caused by unreplicated DNA. Mutants in Cdc20 that were resistant to the spindle checkpoint no longer bound Mad proteins, suggesting that Cdc20 is the target of the spindle checkpoint.
