Association of Rad9 with Double-Strand Breaks through a Mec1-Dependent Mechanism

ABSTRACT Rad9 is required for the activation of DNA damage checkpoint pathways in budding yeast. Rad9 is phosphorylated after DNA damage in a Mec1- and Tel1-dependent manner and subsequently interacts with Rad53. This Rad9-Rad53 interaction has been suggested to trigger the activation and phosphorylation of Rad53. Here we show that Mec1 controls the Rad9 accumulation at double-strand breaks (DSBs). Rad9 was phosphorylated after DSB induction and associated with DSBs. However, its phosphorylation and association with DSBs were significantly decreased in cells carrying a mec1Δ or kinase-negative mec1 mutation. Mec1 phosphorylated the S/TQ motifs of Rad9 in vitro, the same motifs that are phosphorylated after DNA damage in vivo. In addition, multiple mutations in the Rad9 S/TQ motifs resulted in its defective association with DSBs. Phosphorylation of Rad9 was partially defective in cells carrying a weak mec1 allele (mec1-81), whereas its association with DSBs occurred efficiently in the mec1-81 mutants, as found in wild-type cells. However, the Rad9-Rad53 interaction after DSB induction was significantly decreased in mec1-81 mutants, as it was in mec1Δ mutants. Deletion mutation in RAD53 did not affect the association of Rad9 with DSBs. Our results suggest that Mec1 promotes association of Rad9 with sites of DNA damage, thereby leading to full phosphorylation of Rad9 and its interaction with Rad53.

[1]  L. Hartwell,et al.  CDC5 and CKII Control Adaptation to the Yeast DNA Damage Checkpoint , 1997, Cell.

[2]  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.

[3]  R. Abraham.  Cell cycle checkpoint signaling through the ATM and ATR kinases. , 2001, Genes & development.

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

[5]  John Rouse,et al.  Lcd1p recruits Mec1p to DNA lesions in vitro and in vivo. , 2002, Molecular cell.

[6]  Jun Qin,et al.  ATR and ATRIP: Partners in Checkpoint Signaling , 2001, Science.

[7]  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.

[8]  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.

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

[10]  M. Yanagida,et al.  Damage and replication checkpoint control in fission yeast is ensured by interactions of Crb2, a protein with BRCT motif, with Cut5 and Chk1. , 1997, Genes & development.

[11]  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.

[12]  Robert J. D. Reid,et al.  Efficient PCR‐based gene disruption in Saccharomyces strains using intergenic primers , 2002, Yeast.

[13]  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.

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

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

[16]  D. Toczyski,et al.  Two checkpoint complexes are independently recruited to sites of DNA damage in vivo. , 2001, Genes & development.

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

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

[19]  Katsunori Sugimoto,et al.  Pie1, a Protein Interacting with Mec1, Controls Cell Growth and Checkpoint Responses in Saccharomyces cerevisiae , 2001, Molecular and Cellular Biology.

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

[21]  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.

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

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

[24]  Kunihiro Matsumoto,et al.  The ATM-related Tel1 protein of Saccharomyces cerevisiae controls a checkpoint response following phleomycin treatment. , 2003, Nucleic acids research.

[25]  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.

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

[27]  Katsuhiko Shirahige,et al.  S-phase checkpoint proteins Tof1 and Mrc1 form a stable replication-pausing complex , 2003, Nature.

[28]  T. Petes,et al.  Protein kinase activity of Tel1p and Mec1p, two Saccharomyces cerevisiae proteins related to the human ATM protein kinase. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[29]  Kunihiro Matsumoto,et al.  ATM-related Tel1 associates with double-strand breaks through an Xrs2-dependent mechanism. , 2003, Genes & development.

[30]  J. Haber,et al.  Regulation of Saccharomyces Rad53 checkpoint kinase during adaptation from DNA damage-induced G2/M arrest. , 2001, Molecular cell.

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

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

[33]  P. Russell,et al.  Retention but Not Recruitment of Crb2 at Double-Strand Breaks Requires Rad1 and Rad3 Complexes , 2003, Molecular and Cellular Biology.

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

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

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

[37]  Stephen J. Elledge,et al.  Sensing DNA Damage Through ATRIP Recognition of RPA-ssDNA Complexes , 2003, Science.

[38]  Kunihiro Matsumoto,et al.  Recruitment of Mec1 and Ddc1 Checkpoint Proteins to Double-Strand Breaks Through Distinct Mechanisms , 2001, Science.

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

[40]  J. Haber,et al.  Intermediates of recombination during mating type switching in Saccharomyces cerevisiae. , 1990, The EMBO journal.