ABSTRACT
Cohesin mediates sister chromatid cohesion to enable chromosome segregation and DNA damage repair. To perform these functions, cohesin needs to be protected from WAPL, which otherwise releases cohesin from DNA. It has been proposed that cohesin is protected from WAPL by SORORIN. However, in vivo evidence for this antagonism is missing and SORORIN is only known to exist in vertebrates and insects. It is therefore unknown how important and widespread SORORIN's functions are. Here we report the identification of SORORIN orthologs in Schizosaccharomyces pombe (Sor1) and Arabidopsis thaliana (AtSORORIN). sor1Δ mutants display cohesion defects, which are partially alleviated by wpl1Δ. Atsororin mutant plants display dwarfism, tissue specific cohesion defects and chromosome mis-segregation. Furthermore, Atsororin mutant plants are sterile and separate sister chromatids prematurely at anaphase I. The somatic, but not the meiotic deficiencies can be alleviated by loss of WAPL. These results provide in vivo evidence for SORORIN antagonizing WAPL, reveal that SORORIN is present in organisms beyond the animal kingdom and indicate that it has acquired tissue specific functions in plants.
Subject(s)
Arabidopsis Proteins , Arabidopsis , Cell Cycle Proteins , Chromosomal Proteins, Non-Histone , Schizosaccharomyces pombe Proteins , Schizosaccharomyces , Arabidopsis/genetics , Arabidopsis/metabolism , Cell Cycle Proteins/metabolism , Cell Cycle Proteins/genetics , Schizosaccharomyces pombe Proteins/metabolism , Schizosaccharomyces pombe Proteins/genetics , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Chromosomal Proteins, Non-Histone/metabolism , Chromosomal Proteins, Non-Histone/genetics , Schizosaccharomyces/genetics , Schizosaccharomyces/metabolism , Cohesins , Chromosome Segregation , Mutation , Chromatids/metabolism , Chromatids/genetics , Evolution, Molecular , Meiosis/geneticsABSTRACT
One of the key features of meiosis is that shugoshin in complex with protein phosphatase 2A (PP2A) protects centromeric cohesin during meiosis I, but not during meiosis II. A new model suggests that a PP2A inhibitor mediates deprotection of centromeric cohesin during meiosis II.
Subject(s)
Ciona intestinalis/physiology , Meiosis , Protein Phosphatase 2/metabolism , Sister Chromatid Exchange , Transcription Factors/metabolism , Animals , MaleABSTRACT
The reduction of chromosome number during meiosis is achieved by two successive rounds of chromosome segregation after just single round of DNA replication. To identify novel proteins required for the proper segregation of chromosomes during meiosis, we analyzed the consequences of deleting Schizosaccharomyces pombe genes predicted to encode protein kinases that are not essential for cell viability. We show that Mph1, a member of the Mps1 family of spindle assembly checkpoint kinases, is required to prevent meiosis I homolog non-disjunction. We also provide evidence for a novel function of Spo4, the fission yeast ortholog of Dbf4-dependent Cdc7 kinase, in regulating the length of anaphase II spindles. In the absence of Spo4, abnormally elongated anaphase II spindles frequently overlap and thus destroy the linear order of nuclei in the ascus. Our observation that the spo4Δ mutant phenotype can be partially suppressed by inhibiting Cdc2-as suggests that dysregulation of the activity of this cyclin-dependent kinase may cause abnormal elongation of anaphase II spindles in spo4Δ mutant cells.
Subject(s)
Chromosomes, Fungal/genetics , Gene Expression Regulation, Fungal , Meiosis/genetics , Nondisjunction, Genetic , Schizosaccharomyces/genetics , CDC2 Protein Kinase/antagonists & inhibitors , CDC2 Protein Kinase/genetics , CDC2 Protein Kinase/metabolism , Cell Cycle Proteins/deficiency , Cell Cycle Proteins/genetics , Cell Nucleus/genetics , Cell Nucleus/metabolism , Chromosomes, Fungal/ultrastructure , DNA Replication , Gene Knockout Techniques , Genotype , Phenotype , Plasmids/genetics , Protein Kinases/genetics , Protein Kinases/metabolism , Protein Serine-Threonine Kinases/deficiency , Protein Serine-Threonine Kinases/genetics , Schizosaccharomyces/metabolism , Schizosaccharomyces/ultrastructure , Schizosaccharomyces pombe Proteins/antagonists & inhibitors , Schizosaccharomyces pombe Proteins/genetics , Schizosaccharomyces pombe Proteins/metabolismABSTRACT
The genome of the fission yeast Schizosaccharomyces pombe encodes for 17 protein kinases that are essential for viability. Studies of the essential kinases often require the use of mutant strains carrying conditional alleles. To inactivate these kinases conditionally, we applied a recently developed chemical genetic strategy. The mutation of a single residue in the ATP-binding pocket confers sensitivity to small-molecule inhibitors, allowing for specific inactivation of the modified kinase. Using this approach, we constructed conditional analog-sensitive alleles of 13 essential protein kinases in the fission yeast S. pombe.
Subject(s)
Alleles , Models, Biological , Protein Kinases/genetics , Schizosaccharomyces/enzymology , Adenosine Triphosphate/metabolism , Binding Sites/genetics , Cinnamates , DNA Primers/genetics , Escherichia coli , Gene Knockout Techniques , Genes, Essential/genetics , Hygromycin B/analogs & derivatives , Molecular Structure , Mutation/genetics , Protein Kinases/metabolism , Sequence Analysis, DNA , Species Specificity , StreptothricinsABSTRACT
A large number of functional foods, including those that contain ß-d-glucans, have been shown to prevent human DNA against genotoxic effects and associated development of cancer and other chronic diseases. In this paper, carboxymethyl chitin-glucan (CM-CG) isolated from Aspergillus niger was investigated from two standpoints: (1) DNA-protective effects against oxidative DNA damage induced by H(2)O(2) and alkylating DNA damage induced by MMS and MNNG, and (2) a potential effect on rejoining of MMS- and MNNG-induced single strand DNA breaks. The results obtained by the comet assay in human cells cultured in vitro showed that CM-CG reduced significantly the level of oxidative DNA lesions induced by H(2)O(2) but did not change the level of alkylating DNA lesions induced by MMS or MNNG. On the other side, the efficiency of DNA-rejoining of single strand DNA breaks induced by MMS and MNNG was significantly higher in HepG2 cells pre-treated with CM-CG. The antioxidative activity of carboxymethyl chitin-glucan was confirmed by the DPPH assay.
Subject(s)
Chitin/analogs & derivatives , Glucans/pharmacology , Methyl Methanesulfonate/toxicity , Methylnitronitrosoguanidine/toxicity , Oxidative Stress/drug effects , Alkylating Agents/toxicity , Antioxidants/isolation & purification , Antioxidants/pharmacology , Aspergillus niger/chemistry , Chitin/isolation & purification , Chitin/pharmacology , Comet Assay , DNA Breaks, Single-Stranded/drug effects , DNA Damage/drug effects , DNA Repair/drug effects , Glucans/isolation & purification , HeLa Cells , Hep G2 Cells , Humans , Hydrogen Peroxide/toxicityABSTRACT
Meiosis is the process which produces haploid gametes from diploid precursor cells. This reduction of chromosome number is achieved by two successive divisions. Whereas homologs segregate during meiosis I, sister chromatids segregate during meiosis II. To identify novel proteins required for proper segregation of chromosomes during meiosis, we applied a high-throughput knockout technique to delete 87 S. pombe genes whose expression is upregulated during meiosis and analyzed the mutant phenotypes. Using this approach, we identified a new protein, Dil1, which is required to prevent meiosis I homolog non-disjunction. We show that Dil1 acts in the dynein pathway to promote oscillatory nuclear movement during meiosis.