Pie1, a Protein Interacting with Mec1, Controls Cell Growth and Checkpoint Responses in Saccharomyces cerevisiae

ABSTRACT In eukaryotes, the ATM and ATR family proteins play a critical role in the DNA damage and replication checkpoint controls. These proteins are characterized by a kinase domain related to the phosphatidylinositol 3-kinase, but they have the ability to phosphorylate proteins. In budding yeast, the ATR family protein Mec1/Esr1 is essential for checkpoint responses and cell growth. We have isolated the PIE1 gene in a two-hybrid screen for proteins that interact with Mec1, and we show that Pie1 interacts physically with Mec1 in vivo. Like MEC1, PIE1is essential for cell growth, and deletion of the PIE1 gene causes defects in the DNA damage and replication block checkpoints similar to those observed in mec1Δ mutants. Rad53 hyperphosphorylation following DNA damage and replication block is also decreased in pie1Δ cells, as in mec1Δcells. Pie1 has a limited homology to fission yeast Rad26, which forms a complex with the ATR family protein Rad3. Mutation of the region in Pie1 homologous to Rad26 results in a phenotype similar to that of thepie1Δ mutation. Mec1 protein kinase activity appears to be essential for checkpoint responses and cell growth. However, Mec1 kinase activity is unaffected by the pie1Δ mutation, suggesting that Pie1 regulates some essential function other than Mec1 kinase activity. Thus, Pie1 is structurally and functionally related to Rad26 and interacts with Mec1 to control checkpoints and cell proliferation.

[1]  R Rothstein,et al.  A suppressor of two essential checkpoint genes identifies a novel protein that negatively affects dNTP pools. , 1998, Molecular cell.

[2]  L. Hartwell,et al.  Cell cycle arrest of cdc mutants and specificity of the RAD9 checkpoint. , 1993, Genetics.

[3]  D. Stern,et al.  Rad53 FHA domain associated with phosphorylated Rad9 in the DNA damage checkpoint. , 1998, Science.

[4]  V. Zakian.  ATM-related genes: What do they tell us about functions of the human gene? , 1995, Cell.

[5]  G. Lucchini,et al.  The novel DNA damage checkpoint protein Ddc1p is phosphorylated periodically during the cell cycle and in response to DNA damage in budding yeast , 1997, The EMBO journal.

[6]  S. Osmani,et al.  Checkpoint defects leading to premature mitosis also cause endoreplication of DNA in Aspergillus nidulans. , 1999, Molecular biology of the cell.

[7]  G. Lucchini,et al.  Mec1p is essential for phosphorylation of the yeast DNA damage checkpoint protein Ddc1p, which physically interacts with Mec3p , 1998, The EMBO journal.

[8]  S. Jackson,et al.  LCD1: an essential gene involved in checkpoint control and regulation of the MEC1 signalling pathway in Saccharomyces cerevisiae , 2000, The EMBO journal.

[9]  A. Carr,et al.  The Schizosaccharomyces pombe rad3 checkpoint gene. , 1996, The EMBO journal.

[10]  Y Taya,et al.  Activation of the ATM kinase by ionizing radiation and phosphorylation of p53. , 1998, Science.

[11]  M. Lovett,et al.  A single ataxia telangiectasia gene with a product similar to PI-3 kinase. , 1995, Science.

[12]  J. Sekelsky,et al.  mus304 encodes a novel DNA damage checkpoint protein required during Drosophila development. , 2000, Genes & development.

[13]  Kunihiro Matsumoto,et al.  Rfc5, in Cooperation with Rad24, Controls DNA Damage Checkpoints throughout the Cell Cycle inSaccharomyces cerevisiae , 2000, Molecular and Cellular Biology.

[14]  G. Blobel,et al.  Isolation of yeast nuclei. , 1991, Methods in enzymology.

[15]  A. Carr,et al.  Analysis of Rad3 and Chk1 protein kinases defines different checkpoint responses , 1998, The EMBO journal.

[16]  S. Elledge,et al.  Ataxia telangiectasia-mutated phosphorylates Chk2 in vivo and in vitro. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[17]  A. Carr.  Control of cell cycle arrest by the Mec1sc/Rad3sp DNA structure checkpoint pathway. , 1997, Current opinion in genetics & development.

[18]  A. Carr,et al.  A Rad3–Rad26 complex responds to DNA damage independently of other checkpoint proteins , 1999, Nature Cell Biology.

[19]  G. Lucchini,et al.  DNA damage checkpoint in budding yeast , 1998, The EMBO journal.

[20]  A. Emili,et al.  MEC1-dependent phosphorylation of Rad9p in response to DNA damage. , 1998, Molecular cell.

[21]  L. Hartwell,et al.  Checkpoints: controls that ensure the order of cell cycle events. , 1989, Science.

[22]  I. Galli,et al.  A novel mutant allele of Schizosaccharomyces pombe rad26 defective in monitoring S-phase progression to prevent premature mitosis , 1997, Molecular and cellular biology.

[23]  S. Jackson,et al.  DNA-dependent protein kinase. , 1997, The international journal of biochemistry & cell biology.

[24]  S. Elledge,et al.  The SAD1/RAD53 protein kinase controls multiple checkpoints and DNA damage-induced transcription in yeast. , 1994, Genes & development.

[25]  F. Collins,et al.  TEL1, an S. cerevisiae homolog of the human gene mutated in ataxia telangiectasia, is functionally related to the yeast checkpoint gene MEC1 , 1995, Cell.

[26]  L. Hartwell,et al.  Mitotic checkpoint genes in budding yeast and the dependence of mitosis on DNA replication and repair. , 1994, Genes & development.

[27]  Stephen J. Elledge,et al.  Cell Cycle Checkpoints: Preventing an Identity Crisis , 1996, Science.

[28]  H. Ogawa,et al.  An essential gene, ESR1, is required for mitotic cell growth, DNA repair and meiotic recombination in Saccharomyces cerevisiae. , 1994, Nucleic acids research.

[29]  Y Taya,et al.  Enhanced phosphorylation of p53 by ATM in response to DNA damage. , 1998, Science.

[30]  R. D. Gietz,et al.  New yeast-Escherichia coli shuttle vectors constructed with in vitro mutagenized yeast genes lacking six-base pair restriction sites. , 1988, Gene.

[31]  G. Fink,et al.  Methods in yeast genetics , 1979 .

[32]  A. Carr,et al.  Identification and characterization of new elements involved in checkpoint and feedback controls in fission yeast. , 1994, Molecular biology of the cell.

[33]  A. Carr,et al.  The Atr and Atm protein kinases associate with different sites along meiotically pairing chromosomes. , 1996, Genes & development.

[34]  S. Ando,et al.  Functional and Physical Interaction between Rad24 and Rfc5 in the Yeast Checkpoint Pathways , 1998, Molecular and Cellular Biology.

[35]  T. Baker,et al.  Complete transposition requires four active monomers in the mu transposase tetramer. , 1994, Genes & development.

[36]  L. Hartwell,et al.  The RAD9 gene controls the cell cycle response to DNA damage in Saccharomyces cerevisiae. , 1988, Science.

[37]  G. Lucchini,et al.  The checkpoint protein Ddc2, functionally related to S. pombe Rad26, interacts with Mec1 and is regulated by Mec1-dependent phosphorylation in budding yeast. , 2000, Genes & development.

[38]  K. Matsumoto,et al.  Rfc5, a replication factor C component, is required for regulation of Rad53 protein kinase in the yeast checkpoint pathway , 1997, Molecular and cellular biology.

[39]  R. Laskey,et al.  Nuclear targeting sequences--a consensus? , 1991, Trends in biochemical sciences.

[40]  S. Elledge,et al.  Regulation of RAD53 by the ATM-Like Kinases MEC1 and TEL1 in Yeast Cell Cycle Checkpoint Pathways , 1996, Science.

[41]  D. Stern,et al.  Spk1/Rad53 is regulated by Mec1-dependent protein phosphorylation in DNA replication and damage checkpoint pathways. , 1996, Genes & Development.

[42]  S. Elledge,et al.  Recovery from DNA replicational stress is the essential function of the S-phase checkpoint pathway. , 1998, Genes & development.

[43]  E. Craig,et al.  Genomic libraries and a host strain designed for highly efficient two-hybrid selection in yeast. , 1996, Genetics.

[44]  Kunihiro Matsumoto,et al.  Role of a Complex Containing Rad17, Mec3, and Ddc1 in the Yeast DNA Damage Checkpoint Pathway , 1999, Molecular and Cellular Biology.

[45]  H. Erdjument-Bromage,et al.  A novel Rad24 checkpoint protein complex closely related to replication factor C , 2000, Current Biology.

[46]  J. Sekelsky,et al.  The mei-41 gene of D. melanogaster is a structural and functional homolog of the human ataxia telangiectasia gene , 1995, Cell.

[47]  J. Piette,et al.  Non‐enzymatic triggering of the ceramide signalling cascade by solar UVA radiation , 2000, The EMBO journal.

[48]  D. Lydall,et al.  Yeast Checkpoint Genes in DNA Damage Processing: Implications for Repair and Arrest , 1995, Science.

[49]  Janina Maier,et al.  Guide to yeast genetics and molecular biology. , 1991, Methods in enzymology.

[50]  J. Vialard,et al.  The budding yeast Rad9 checkpoint protein is subjected to Mec1/Tel1‐dependent hyperphosphorylation and interacts with Rad53 after DNA damage , 1998, The EMBO journal.

[51]  H. Wang,et al.  Control of the DNA damage checkpoint by chk1 and rad53 protein kinases through distinct mechanisms. , 1999, Science.

[52]  G. Blobel,et al.  Yeast nuclear envelope proteins cross react with an antibody against mammalian pore complex proteins , 1989, The Journal of cell biology.