A novel genetic screen identifies checkpoint-defective alleles of Schizosaccharomyces pombe chk1.
The protein kinase Chk1 is required in the fission yeast Schizosaccharomyces pombe for delaying cell cycle progression in response to DNA damage. Chk1 becomes phosphorylated when DNA is damaged by a variety of agents, including the anti-tumor drug camptothecin. To further characterize the behavior of Chk1 in response to DNA damage, we used PCR-based mutagenesis of the chk1 gene coupled with in vivo gap repair to generate mutant alleles. Of 44 chk1 mutants recovered, six encode full-length proteins that confer a DNA damage-sensitive phenotype. All of the alleles render cells checkpoint-defective, but confer subtle differences in sensitivity to camptothecin or UV light. Mutant alleles were sequenced and served to identify regions of the protein that are critical for checkpoint function.
Department of Pharmacology, UMDNJ-Robert Wood Johnson Medical School, Piscataway, NJ 08854-5635, USA.
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Wan
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Walworth
D000483:Alleles; D000595:Amino Acid Sequence; D002166:Camptothecin / Q000494:pharmacology; D018797:Cell Cycle Proteins; D000071877:Checkpoint Kinase 1; D017124:Conserved Sequence; D004249:DNA Damage; D004261:DNA Replication; D005800:Genes, Fungal; D018816:Genes, cdc; D005838:Genotype; D006918:Hydroxyurea / Q000494:pharmacology; D008969:Molecular Sequence Data; D009154:Mutation; D009687:Nuclear Proteins; D010641:Phenotype; D010766:Phosphorylation; D017354:Point Mutation / Q000187:drug effects*; D011494:Protein Kinases / Q000235:genetics*; D011505:Protein-Tyrosine Kinases / Q000235:genetics; D012568:Schizosaccharomyces / Q000235:genetics*; D029702:Schizosaccharomyces pombe Proteins; D014466:Ultraviolet Rays
D018797:Cell Cycle Proteins; D009687:Nuclear Proteins; D029702:Schizosaccharomyces pombe Proteins; D011494:Protein Kinases; C068689:wee1 protein, S pombe; D011505:Protein-Tyrosine Kinases; D000071877:Checkpoint Kinase 1; C000605873:Chk1 protein, S pombe; D006918:Hydroxyurea; D002166:Camptothecin