Mutations in SPK1/RAD53 that specifically abolish checkpoint but not growth-related functions.
SPK1/RAD53/SAD1/MEC2 encodes an essential protein kinase of Saccharomyces cerevisiae and is required for the execution of checkpoint arrest at multiple stages of the cell cycle. We have isolated two mutant alleles of SPK1 (spk1K227A and spk1-1A208P) that are defective for checkpoint-arrest functions but retain wild-type levels of SPK1-associated growth activity. Both mutations occur within conserved regions of the kinase domain of SPK1 resulting in a substantial reduction in the catalytic activity of Spk1. Thus, while minimal levels of Spk1 kinase activity are capable of supporting normal rates of growth, higher levels are required for checkpoint functions. In addition, using deletional analysis we have identified a region within the N-terminus of Spk1 outside of the conserved kinase domain that is required for checkpoint functions. Interestingly, this region may be important in the regulation of Spk1 kinase activity.
Fay
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Department of Pathology, Yale University School of Medicine, New Haven, CT 06520-8023, USA.
Sun
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Stern
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D000483:Alleles; D018797:Cell Cycle Proteins; D064447:Checkpoint Kinase 2; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D015971:Gene Expression Regulation, Enzymologic; D015966:Gene Expression Regulation, Fungal; D016296:Mutagenesis; D010957:Plasmids; D011494:Protein Kinases / Q000235:genetics* / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D029701:Saccharomyces cerevisiae Proteins; D017384:Sequence Deletion; D014170:Transformation, Genetic
D018797:Cell Cycle Proteins; D029701:Saccharomyces cerevisiae Proteins; D011494:Protein Kinases; D064447:Checkpoint Kinase 2; D017346:Protein Serine-Threonine Kinases; C067022:RAD53 protein, S cerevisiae