Pds1 phosphorylation in response to DNA damage is essential for its DNA damage checkpoint function.

In Saccharomyces cerevisiae, Pds1 is an anaphase inhibitor and plays an essential role in DNA damage and spindle checkpoint pathways. Pds1 is phosphorylated in response to DNA damage but not spindle disruption, indicating distinct mechanisms delaying anaphase entry. Phosphorylation of Pds1 is Mec1 and Chk1 dependent in vivo. Here, we show that Pds1 is phosphorylated at multiple sites in vivo in response to DNA damage by Chk1. Mutation of the Chk1 phosphorylation sites on Pds1 abolished most of its DNA damage-inducible phosphorylation and its checkpoint function, whereas its anaphase inhibitor functions and spindle checkpoint functions remain intact. Loss of Pds1 phosphorylation correlates with APC-dependent Pds1 destruction in response to DNA damage. We also show that APC(Cdc20) is active in preanaphase arrested cells after DNA damage. This suggests that Pds1 is stabilized by phosphorylation in response to DNA damage, but APC(Cdc20) activity is not altered. Our results indicate that phosphorylation of Pds1 by Chk1 is the key function of Chk1 required to prevent anaphase entry.

[1]  J. Murguía,et al.  Sensing and responding to DNA damage. , 2000, Current opinion in genetics & development.

[2]  K Nasmyth,et al.  Control of cyclin ubiquitination by CDK-regulated binding of Hct1 to the anaphase promoting complex. , 1998, Science.

[3]  D. Koshland,et al.  Pds1p of budding yeast has dual roles: inhibition of anaphase initiation and regulation of mitotic exit. , 1999, Genes & development.

[4]  David O. Morgan,et al.  Regulation of the APC and the exit from mitosis , 1999, Nature Cell Biology.

[5]  M. Kirschner,et al.  Identification of a vertebrate sister-chromatid separation inhibitor involved in transformation and tumorigenesis. , 1999, Science.

[6]  M. Kirschner,et al.  Anaphase initiation in Saccharomyces cerevisiae is controlled by the APC-dependent degradation of the anaphase inhibitor Pds1p. , 1996, Genes & development.

[7]  S. Prinz,et al.  CDC20 and CDH1: a family of substrate-specific activators of APC-dependent proteolysis. , 1997, Science.

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

[9]  N. Walworth,et al.  The G2-phase DNA-damage checkpoint. , 2000, Trends in cell biology.

[10]  S. Elledge,et al.  Conservation of the Chk1 checkpoint pathway in mammals: linkage of DNA damage to Cdk regulation through Cdc25. , 1997, Science.

[11]  Kim Nasmyth,et al.  The Polo‐like kinase Cdc5p and the WD‐repeat protein Cdc20p/fizzy are regulators and substrates of the anaphase promoting complex in Saccharomyces cerevisiae , 1998, The EMBO journal.

[12]  T. Weinert.  A DNA Damage Checkpoint Meets the Cell Cycle Engine , 1997, Science.

[13]  K Nasmyth,et al.  TPR proteins required for anaphase progression mediate ubiquitination of mitotic B-type cyclins in yeast. , 1996, Molecular biology of the cell.

[14]  Andrew W. Murray,et al.  Association of Spindle Assembly Checkpoint Component XMAD2 with Unattached Kinetochores , 1996, Science.

[15]  M. Karin,et al.  Phosphorylation meets ubiquitination: the control of NF-[kappa]B activity. , 2000, Annual review of immunology.

[16]  N. Rhind,et al.  Cdc2 tyrosine phosphorylation is required for the DNA damage checkpoint in fission yeast. , 1997, Genes & development.

[17]  C. Peng,et al.  Mitotic and G2 checkpoint control: regulation of 14-3-3 protein binding by phosphorylation of Cdc25C on serine-216. , 1997, Science.

[18]  Michael Schwab,et al.  Yeast Hct1 Is a Regulator of Clb2 Cyclin Proteolysis , 1997, Cell.

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

[20]  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.

[21]  K. Nasmyth,et al.  Cohesins: Chromosomal Proteins that Prevent Premature Separation of Sister Chromatids , 1997, Cell.

[22]  S. Elledge,et al.  How the Cyclin Became a Cyclin Regulated Proteolysis in the Cell Cycle , 1999, Cell.

[23]  A. Straight.  Cell cycle: Checkpoint proteins and kinetochores , 1997, Current Biology.

[24]  M. Yanagida,et al.  The fission yeast cut1 + gene regulates spindle pole body duplication and has homology to the budding yeast ESP1 gene , 1990, Cell.

[25]  Stuart Tugendreich,et al.  CDC27Hs colocalizes with CDC16Hs to the centrosome and mitotic spindle and is essential for the metaphase to anaphase transition , 1995, Cell.

[26]  N. Rhind,et al.  Chk1 and Cds1: linchpins of the DNA damage and replication checkpoint pathways. , 2000, Journal of cell science.

[27]  D. Morgan,et al.  Pds1 and Esp1 control both anaphase and mitotic exit in normal cells and after DNA damage. , 1999, Genes & development.

[28]  P. Russell,et al.  Cdc25 inhibited in vivo and in vitro by checkpoint kinases Cds1 and Chk1. , 1999, Molecular biology of the cell.

[29]  Attila Tóth,et al.  APCCdc20 promotes exit from mitosis by destroying the anaphase inhibitor Pds1 and cyclin Clb5 , 1999, Nature.

[30]  N. Rhind,et al.  Cdc25 mitotic inducer targeted by chk1 DNA damage checkpoint kinase. , 1997, Science.

[31]  Stephen J. Elledge,et al.  The SCFβ-TRCP–ubiquitin ligase complex associates specifically with phosphorylated destruction motifs in IκBα and β-catenin and stimulates IκBα ubiquitination in vitro , 1999 .

[32]  J. R. Hutchins,et al.  Substrate specificity determinants of the checkpoint protein kinase Chk1 , 2000, FEBS letters.

[33]  H. Wang,et al.  DRC1, DNA replication and checkpoint protein 1, functions with DPB11 to control DNA replication and the S-phase checkpoint in Saccharomyces cerevisiae. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[34]  D. J. Clarke,et al.  A Novel Role of the Budding Yeast Separin Esp1 in Anaphase Spindle Elongation , 2001, The Journal of cell biology.

[35]  Friedrich Lottspeich,et al.  Sister-chromatid separation at anaphase onset is promoted by cleavage of the cohesin subunit Scc1 , 1999, Nature.

[36]  A. Hershko.  Roles of ubiquitin-mediated proteolysis in cell cycle control. , 1997, Current opinion in cell biology.

[37]  M. Kirschner,et al.  A 20s complex containing CDC27 and CDC16 catalyzes the mitosis-specific conjugation of ubiquitin to cyclin B , 1995, Cell.

[38]  A. Murray.  Cyclin Ubiquitination: The destructive end of mitosis , 1995, Cell.

[39]  Tim Hunt,et al.  Cut2 proteolysis required for sister-chromatid separation in fission yeast , 1996, Nature.

[40]  P. Beer-Romero,et al.  SCFβ-TRCP and phosphorylation dependent ubiquitination of IκBα catalyzed by Ubc3 and Ubc4 , 2000, Oncogene.

[41]  D. Koshland,et al.  The anaphase inhibitor of Saccharomyces cerevisiae Pds1p is a target of the DNA damage checkpoint pathway. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[42]  S. Elledge,et al.  The DNA damage response: putting checkpoints in perspective , 2000, Nature.

[43]  W. Krek,et al.  Proteolysis and the G1-S transition: the SCF connection. , 1998, Current opinion in genetics & development.

[44]  P. Silver,et al.  Dynamic localization of the nuclear import receptor and its interactions with transport factors , 1996, The Journal of cell biology.

[45]  D. J. Clarke,et al.  Dosage Suppressors of pds1 Implicate Ubiquitin-Associated Domains in Checkpoint Control , 2001, Molecular and Cellular Biology.

[46]  R. Sikorski,et al.  A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. , 1989, Genetics.

[47]  A. Hershko,et al.  Reversible phosphorylation controls the activity of cyclosome-associated cyclin-ubiquitin ligase. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[48]  M. Kirschner,et al.  The checkpoint protein MAD2 and the mitotic regulator CDC20 form a ternary complex with the anaphase-promoting complex to control anaphase initiation. , 1998, Genes & development.

[49]  David O. Morgan,et al.  The Polo-related kinase Cdc5 activates and is destroyed by the mitotic cyclin destruction machinery in S. cerevisiae , 1998, Current Biology.

[50]  V. Guacci,et al.  Pds1p, an inhibitor of anaphase in budding yeast, plays a critical role in the APC and checkpoint pathway(s) , 1996, The Journal of cell biology.

[51]  P. Nurse,et al.  Chk1 is a wee1 kinase in the G2 DNA damage checkpoint inhibiting cdc2 by Y15 phosphorylation , 1997, The EMBO journal.

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

[53]  R. Benezra,et al.  MAD2 associates with the cyclosome/anaphase-promoting complex and inhibits its activity. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[54]  M. Yanagida,et al.  Fission yeast Cut1 and Cut2 are essential for sister chromatid separation, concentrate along the metaphase spindle and form large complexes. , 1996, The EMBO journal.

[55]  T. Weinert,et al.  RAD53, DUN1 and PDS1 define two parallel G2/M checkpoint pathways in budding yeast , 1999, The EMBO journal.

[56]  Kim Nasmyth,et al.  An ESP1/PDS1 Complex Regulates Loss of Sister Chromatid Cohesion at the Metaphase to Anaphase Transition in Yeast , 1998, Cell.

[57]  Mike Tyers,et al.  F-Box Proteins Are Receptors that Recruit Phosphorylated Substrates to the SCF Ubiquitin-Ligase Complex , 1997, Cell.

[58]  A. Amon.  The spindle checkpoint. , 1999, Current opinion in genetics & development.

[59]  A. Hershko,et al.  The cyclosome, a large complex containing cyclin-selective ubiquitin ligase activity, targets cyclins for destruction at the end of mitosis. , 1995, Molecular biology of the cell.

[60]  L. Hartwell,et al.  Single-stranded DNA arising at telomeres in cdc13 mutants may constitute a specific signal for the RAD9 checkpoint , 1995, Molecular and cellular biology.

[61]  A. Murray,et al.  Budding yeast Cdc20: a target of the spindle checkpoint. , 1998, Science.

[62]  N. Walworth,et al.  Association of Chk1 with 14-3-3 proteins is stimulated by DNA damage. , 1999, Genes & development.

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

[64]  Kim Nasmyth,et al.  Cleavage of Cohesin by the CD Clan Protease Separin Triggers Anaphase in Yeast , 2000, Cell.