An FHA domain–mediated protein interaction network of Rad53 reveals its role in polarized cell growth

The DNA damage checkpoint kinase Rad53 is important for the survival of budding yeast under genotoxic stresses. We performed a biochemical screen to identify proteins with specific affinity for the two Forkhead associated (FHA) domains of Rad53. The N-terminal FHA1 domain was found to coordinate a complex protein interaction network, which includes nuclear proteins involved in DNA damage checkpoints and transcriptional regulation. Unexpectedly, cytosolic proteins involved in cytokinesis, including septins, were also found as FHA1 binding proteins. Consistent with this interaction, a Rad53 mutant defective in its nuclear localization was found to localize to the bud neck. Abnormal morphology was observed in cells overexpressing the FHA1 domain and in rad53Δ cells under DNA replication stress. Further, septin Shs1 appears to have an important role in the response to DNA replication stress. Collectively, the results suggest a novel function of Rad53 in the regulation of polarized cell growth in response to DNA replication stress.

[1]  Marcus B. Smolka,et al.  Checkpoint proteins control morphogenetic events during DNA replication stress in Saccharomyces cerevisiae , 2006, The Journal of cell biology.

[2]  Marcus B Smolka,et al.  Dynamic Changes in Protein-Protein Interaction and Protein Phosphorylation Probed with Amine-reactive Isotope Tag*S , 2005, Molecular & Cellular Proteomics.

[3]  J. Thorner,et al.  Some assembly required: yeast septins provide the instruction manual. , 2005, Trends in cell biology.

[4]  Amy S. Gladfelter,et al.  Interplay between septin organization, cell cycle and cell shape in yeast , 2005, Journal of Cell Science.

[5]  S. Gasser,et al.  Mechanistically distinct roles for Sgs1p in checkpoint activation and replication fork maintenance , 2005, The EMBO journal.

[6]  U. Surana,et al.  DNA replication checkpoint prevents precocious chromosome segregation by regulating spindle behavior. , 2004, Molecular cell.

[7]  P. Russell,et al.  The DNA damage response: sensing and signaling. , 2004, Current opinion in cell biology.

[8]  K. Myung,et al.  Mutator genes for suppression of gross chromosomal rearrangements identified by a genome-wide screening in Saccharomyces cerevisiae , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[9]  M. Tsai,et al.  Mdt1, a Novel Rad53 FHA1 Domain-Interacting Protein, Modulates DNA Damage Tolerance and G2/M Cell Cycle Progression in Saccharomycescerevisiae , 2004, Molecular and Cellular Biology.

[10]  Grant W. Brown,et al.  Cdc7 kinases (DDKs) and checkpoint responses: lessons from two yeasts. , 2003, Mutation research.

[11]  A. Gunjan,et al.  A Rad53 Kinase-Dependent Surveillance Mechanism that Regulates Histone Protein Levels in S. cerevisiae , 2003, Cell.

[12]  M. Tsai,et al.  Diverse but Overlapping Functions of the Two Forkhead-associated (FHA) Domains in Rad53 Checkpoint Kinase Activation* , 2003, Journal of Biological Chemistry.

[13]  M. Longtine,et al.  Regulation of septin organization and function in yeast. , 2003, Trends in cell biology.

[14]  S. Elledge,et al.  Mrc1 is a replication fork component whose phosphorylation in response to DNA replication stress activates Rad53. , 2003, Genes & development.

[15]  J. Diffley,et al.  A central role for DNA replication forks in checkpoint activation and response. , 2003, Molecular cell.

[16]  D. Stern,et al.  FHA Domain-Mediated DNA Checkpoint Regulation of Rad53 , 2003, Cell cycle.

[17]  L. Breeden,et al.  Rad53 Checkpoint Kinase Phosphorylation Site Preference Identified in the Swi6 Protein of Saccharomyces cerevisiae , 2003, Molecular and Cellular Biology.

[18]  J. Haber,et al.  PP2C phosphatases Ptc2 and Ptc3 are required for DNA checkpoint inactivation after a double-strand break. , 2003, Molecular cell.

[19]  Wolf-Dietrich Heyer,et al.  Direct Kinase-to-Kinase Signaling Mediated by the FHA Phosphoprotein Recognition Domain of the Dun1 DNA Damage Checkpoint Kinase , 2003, Molecular and Cellular Biology.

[20]  R. Sternglanz,et al.  Esc1, a Nuclear Periphery Protein Required for Sir4-Based Plasmid Anchoring and Partitioning , 2002, Molecular and Cellular Biology.

[21]  T. Weinert,et al.  Toward maintaining the genome: DNA damage and replication checkpoints. , 2002, Annual review of genetics.

[22]  Monika Tsai-Pflugfelder,et al.  An N-terminal domain of Dbf4p mediates interaction with both origin recognition complex (ORC) and Rad53p and can deregulate late origin firing , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[23]  John R. Yates,et al.  Phospho-Regulation of Kinetochore-Microtubule Attachments by the Aurora Kinase Ipl1p , 2002, Cell.

[24]  S. Jackson,et al.  Interfaces Between the Detection, Signaling, and Repair of DNA Damage , 2002, Science.

[25]  Kyungjae Myung,et al.  Maintenance of Genome Stability in Saccharomyces cerevisiae , 2002, Science.

[26]  M. Snyder,et al.  Microtubule capture by the cleavage apparatus is required for proper spindle positioning in yeast. , 2002, Genes & development.

[27]  D. Stern,et al.  Rad9 phosphorylation sites couple Rad53 to the Saccharomyces cerevisiae DNA damage checkpoint. , 2002, Molecular cell.

[28]  Rodney Rothstein,et al.  The Dun1 checkpoint kinase phosphorylates and regulates the ribonucleotide reductase inhibitor Sml1 , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[29]  Daniel Durocher,et al.  The FHA domain , 2002, FEBS letters.

[30]  P. Bork,et al.  Functional organization of the yeast proteome by systematic analysis of protein complexes , 2002, Nature.

[31]  Gary D Bader,et al.  Systematic identification of protein complexes in Saccharomyces cerevisiae by mass spectrometry , 2002, Nature.

[32]  J. Pringle,et al.  The septin cortex at the yeast mother-bud neck. , 2001, Current opinion in microbiology.

[33]  Stephen J. Elledge,et al.  Mrc1 transduces signals of DNA replication stress to activate Rad53 , 2001, Nature Cell Biology.

[34]  C. Newlon,et al.  The DNA replication checkpoint response stabilizes stalled replication forks , 2001, Nature.

[35]  C. Gilbert,et al.  Budding yeast Rad9 is an ATP-dependent Rad53 activating machine. , 2001, Molecular cell.

[36]  S. Elledge,et al.  Asf1 links Rad53 to control of chromatin assembly. , 2001, Genes & development.

[37]  K. Gould,et al.  Vectors and gene targeting modules for tandem affinity purification in Schizosaccharomyces pombe , 2001, Yeast.

[38]  K. Bloom,et al.  Control of spindle polarity and orientation in Saccharomyces cerevisiae. , 2001, Trends in cell biology.

[39]  N. Lowndes,et al.  DUN1 defines one branch downstream of RAD53 for transcription and DNA damage repair in Saccharomyces cerevisiae , 2000, FEBS letters.

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

[41]  D. Durocher,et al.  The molecular basis of FHA domain:phosphopeptide binding specificity and implications for phospho-dependent signaling mechanisms. , 2000, Molecular cell.

[42]  M. Falconi,et al.  DNA damage checkpoints and DNA replication controls in Saccharomyces cerevisiae. , 2000, Mutation research.

[43]  Chandra L. Theesfeld,et al.  Septin-Dependent Assembly of a Cell Cycle-Regulatory Module in Saccharomyces cerevisiae , 2000, Molecular and Cellular Biology.

[44]  P. Plevani,et al.  Activation of Rad53 kinase in response to DNA damage and its effect in modulating phosphorylation of the lagging strand DNA polymerase , 1999, The EMBO journal.

[45]  D. Kellogg,et al.  Septins: cytoskeletal polymers or signalling GTPases? , 1999, Trends in cell biology.

[46]  D. Durocher,et al.  The FHA domain is a modular phosphopeptide recognition motif. , 1999, Molecular cell.

[47]  Anna Shevchenko,et al.  Exit from Mitosis Is Triggered by Tem1-Dependent Release of the Protein Phosphatase Cdc14 from Nucleolar RENT Complex , 1999, Cell.

[48]  A. Straight,et al.  Net1, a Sir2-Associated Nucleolar Protein Required for rDNA Silencing and Nucleolar Integrity , 1999, Cell.

[49]  M. Snyder,et al.  Nim1-related kinases coordinate cell cycle progression with the organization of the peripheral cytoskeleton in yeast. , 1999, Genes & development.

[50]  K. Tanaka,et al.  Shs1p: a novel member of septin that interacts with spa2p, involved in polarized growth in saccharomyces cerevisiae. , 1998, Biochemical and biophysical research communications.

[51]  K. Shirahige,et al.  Regulation of DNA-replication origins during cell-cycle progression , 1998, Nature.

[52]  J. Diffley,et al.  A Mec1- and Rad53-dependent checkpoint controls late-firing origins of DNA replication , 1998, Nature.

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

[54]  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.

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

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

[57]  P. Philippsen,et al.  Additional modules for versatile and economical PCR‐based gene deletion and modification in Saccharomyces cerevisiae , 1998, Yeast.

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

[59]  S. Jackson,et al.  The recognition of DNA damage. , 1996, Current opinion in genetics & development.

[60]  L. Hartwell,et al.  A checkpoint regulates the rate of progression through S phase in S. cerevisiae in Response to DNA damage , 1995, Cell.

[61]  P Bucher,et al.  The FHA domain: a putative nuclear signalling domain found in protein kinases and transcription factors. , 1995, Trends in biochemical sciences.

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

[63]  K. Nasmyth,et al.  Cell cycle regulated transcription in yeast. , 1994, Current opinion in cell biology.

[64]  L. Hartwell,et al.  Genetic control of the cell division cycle in yeast. , 1974, Science.

[65]  L. Hartwell.  Genetic control of the cell division cycle in yeast. IV. Genes controlling bud emergence and cytokinesis. , 1971, Experimental cell research.

[66]  L. Hartwell,et al.  Dynamic interaction of DNA damage checkpoint protein Rad53 with chromatin assembly factor Asf1. , 2001, Molecular cell.

[67]  J. Thevelein,et al.  The RAS-adenylate cyclase pathway and cell cycle control in , 1992 .