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The novel murine calmodulin-binding protein Sha1 disrupts mitotic spindle and replication checkpoint functions in
1Imperial Cancer Research Fund, Molecular Oncology Laboratory, University of Oxford Institute of Molecular Medicine, John Radcliffe Hospital, Oxford OX3 9DS, UK.
Abstract:
Entry into mitosis is normally blocked in eukaryotic cells that have not completed replicative DNA synthesis; this 'S-M' checkpoint control is fundamental to the maintenance of genomic integrity. Mutants of the fission yeast Schizosaccharomyces pombe defective in the S-M checkpoint fail to arrest the cell cycle when DNA replication is inhibited and hence attempt mitosis and cell division with unreplicated chromosomes, resulting in the 'cut' phenotype. In an attempt to identify conserved molecules involved in the S-M checkpoint we have screened a regulatable murine cDNA library in S. pombe and have identified cDNAs that induce the cut phenotype in cells arrested in S phase by hydroxyurea. One such cDNA encodes a novel protein with multiple calmodulin-binding motifs that, in addition to its effects on the S-M checkpoint, perturbed mitotic spindle functions, although spindle pole duplication was apparently normal. Both aspects of the phenotype induced by this cDNA product, which we term Sha1 (for spindle and hydroxyurea checkpoint abnormal), were suppressed by simultaneous overexpression of calmodulin. Sha1 is structurally related to the product of the Drosophila gene abnormal spindle (asp). These data suggest that calmodulin-binding protein(s) are important in the co-ordination of mitotic spindle functions with mitotic entry in fission yeast, and probably also in multicellular eukaryotes.
Insights
Researchers identified a novel protein, Sha1, crucial for the S-M cell-cycle checkpoint. This protein regulates entry into mitosis and mitotic spindle functions, ensuring genomic integrity.
Area of Science:
- Cell Biology
- Genetics
- Molecular Biology
Background:
- The S-M checkpoint prevents cell division before DNA replication completion, maintaining genomic integrity.
- Defects in this checkpoint lead to attempted mitosis with unreplicated chromosomes, causing the 'cut' phenotype in fission yeast.
Purpose of the Study:
- To identify conserved molecules involved in the S-M checkpoint.
- To investigate the function of novel proteins affecting cell cycle control and mitotic spindle integrity.
Main Methods:
- Screening a murine cDNA library in Schizosaccharomyces pombe.
- Inducing the 'cut' phenotype in hydroxyurea-arrested cells.
- Analyzing the effects of a novel cDNA product (Sha1) on cell cycle progression and spindle function.
- Investigating the role of calmodulin through overexpression studies.
Main Results:
- A novel cDNA encoding a calmodulin-binding protein, Sha1, was identified.
- Sha1 perturbs both the S-M checkpoint and mitotic spindle functions.
- Overexpression of calmodulin suppressed the phenotypes induced by Sha1.
- Sha1 is structurally related to Drosophila's abnormal spindle (asp) protein.
Conclusions:
- Calmodulin-binding proteins are essential for coordinating mitotic spindle functions with mitotic entry.
- These findings suggest conserved roles for calmodulin-binding proteins in cell cycle regulation across eukaryotes.