ABSTRACT
The back-to-back geometry of sister kinetochores is essential in preventing loss or damage of chromosomes during mitosis. Kinetochore orientation is generated in part by a process of resolving kinetochores at the centromere (centromere resolution) prior to spindle interactions. Because few of the genes required for centromere resolution are known, we used Caenorhabditis elegans to screen for conditional mutants defective in orienting sister kinetochores during mitosis. C. elegans is ideal for such screens because its chromosomes are holocentric. Here we identified an essential gene, cin-4, required for centromere resolution and for removal of cohesin from sites near sister kinetochores during mitosis. Given that compromised cohesin function restores centromere resolution in the absence of cin-4, CIN-4 likely acts to remove cohesin from the CENP-A chromatin enabling centromere resolution. CIN-4 has a high amino acid identity to the catalytic domain of topoisomerase II, suggesting a partial gene duplication of the C. elegans topoisomerase II gene, top-2. Similar to CIN-4, TOP-2 is also required for centromere resolution; however, the loss of TOP-2 is phenotypically distinct from the loss of CIN-4, suggesting that CIN-4 and TOP-2 are topoisomerase II isoforms that perform separate essential functions in centromere structure and function.
Subject(s)
Caenorhabditis elegans Proteins/metabolism , Caenorhabditis elegans/cytology , Caenorhabditis elegans/enzymology , Cell Cycle Proteins/metabolism , Chromosomal Proteins, Non-Histone/metabolism , DNA Topoisomerases, Type II/metabolism , Kinetochores/metabolism , Mitosis , Nuclear Proteins/metabolism , Amino Acid Sequence , Animals , Base Sequence , Caenorhabditis elegans/genetics , Caenorhabditis elegans Proteins/chemistry , Caenorhabditis elegans Proteins/genetics , Catalytic Domain , Chromosomal Instability , Chromosomes/metabolism , DNA Topoisomerases, Type II/chemistry , DNA Topoisomerases, Type II/genetics , Embryo, Nonmammalian/cytology , Embryo, Nonmammalian/metabolism , Gene Duplication , Genes, Helminth , Models, Genetic , Molecular Sequence Data , Mutation/genetics , Open Reading Frames/genetics , Protein Transport , RNA Interference , Sequence Homology, Amino Acid , Sequence Homology, Nucleic Acid , CohesinsABSTRACT
Prior to microtubule capture, sister centromeres resolve from one another, coming to rest on opposite surfaces of the condensing chromosome. Subsequent assembly of sister kinetochores at each sister centromere generates a geometry favorable for equal levels of segregation of chromatids. The holocentric chromosomes of Caenorhabditis elegans are uniquely suited for the study of centromere resolution and subsequent kinetochore assembly. In C. elegans, only two proteins have been identified as being necessary for centromere resolution, the kinase AIR-2 (prophase only) and the centromere protein HCP-4/CENP-C. Here we found that the loss of proteins involved in chromosome cohesion bypassed the requirement for HCP-4/CENP-C but not for AIR-2. Interestingly, the loss of cohesin proteins also restored the localization of HCP-6 to the kinetochore. The loss of the condensin II protein HCP-6 or MIX-1/SMC2 impaired centromere resolution. Furthermore, the loss of HCP-6 or MIX-1/SMC2 resulted in no centromere resolution when either nocodazole or RNA interference (RNAi) of the kinetochore protein KNL-1 perturbed spindle-kinetochore interactions. This result suggests that normal prophase centromere resolution is mediated by condensin II proteins, which are actively recruited to sister centromeres to mediate the process of resolution.
