Ddc2 Mediates Mec1 Activation through a Ddc1- or Dpb11-Independent Mechanism

The protein kinase Mec1 (ATR ortholog) and its partner Ddc2 (ATRIP ortholog) play a key role in DNA damage checkpoint responses in budding yeast. Previous studies have established the model in which Ddc1, a subunit of the checkpoint clamp, and Dpb11, related to TopBP1, activate Mec1 directly and control DNA damage checkpoint responses at G1 and G2/M. In this study, we show that Ddc2 contributes to Mec1 activation through a Ddc1- or Dpb11-independent mechanism. The catalytic activity of Mec1 increases after DNA damage in a Ddc2-dependent manner. In contrast, Mec1 activation occurs even in the absence of Ddc1 and Dpb11 function at G2/M. Ddc2 recruits Mec1 to sites of DNA damage. To dissect the role of Ddc2 in Mec1 activation, we isolated and characterized a separation-of-function mutation in DDC2, called ddc2-S4. The ddc2-S4 mutation does not affect Mec1 recruitment but diminishes Mec1 activation. Mec1 phosphorylates histone H2A in response to DNA damage. The ddc2-S4 mutation decreases phosphorylation of histone H2A more significantly than the absence of Ddc1 and Dpb11 function does. Our results suggest that Ddc2 plays a critical role in Mec1 activation as well as Mec1 localization at sites of DNA damage.

[1]  Katsunori Sugimoto,et al.  Association of Rad9 with Double-Strand Breaks through a Mec1-Dependent Mechanism , 2004, Molecular and Cellular Biology.

[2]  M. Wold.  Replication protein A: a heterotrimeric, single-stranded DNA-binding protein required for eukaryotic DNA metabolism. , 1997, Annual review of biochemistry.

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

[4]  J Wade Harper,et al.  The DNA damage response: ten years after. , 2007, Molecular cell.

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

[6]  Joon-Kyu Lee,et al.  Phosphorylation of Chk1 by ATM- and Rad3-related (ATR) in Xenopus Egg Extracts Requires Binding of ATRIP to ATR but Not the Stable DNA-binding or Coiled-coil Domains of ATRIP* , 2005, Journal of Biological Chemistry.

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

[8]  P. Plevani,et al.  Dynamics of Rad9 Chromatin Binding and Checkpoint Function Are Mediated by Its Dimerization and Are Cell Cycle–Regulated by CDK1 Activity , 2010, PLoS genetics.

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

[10]  P. Plevani,et al.  Sensing of Replication Stress and Mec1 Activation Act through Two Independent Pathways Involving the 9-1-1 Complex and DNA Polymerase ε , 2011, PLoS genetics.

[11]  J. Myers,et al.  ATRIP binding to replication protein A-single-stranded DNA promotes ATR-ATRIP localization but is dispensable for Chk1 phosphorylation. , 2005, Molecular biology of the cell.

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

[13]  Jerzy Majka,et al.  Yeast Rad17/Mec3/Ddc1: A sliding clamp for the DNA damage checkpoint , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[14]  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.

[15]  Katsunori Sugimoto,et al.  Role of the C terminus of Mec1 checkpoint kinase in its localization to sites of DNA damage. , 2005, Molecular biology of the cell.

[16]  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.

[17]  P. Philippsen,et al.  New heterologous modules for classical or PCR‐based gene disruptions in Saccharomyces cerevisiae , 1994, Yeast.

[18]  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.

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

[20]  James E Haber,et al.  Surviving the breakup: the DNA damage checkpoint. , 2006, Annual review of genetics.

[21]  Paolo Plevani,et al.  The DNA Damage Checkpoint Response Requires Histone H2B Ubiquitination by Rad6-Bre1 and H3 Methylation by Dot1* , 2005, Journal of Biological Chemistry.

[22]  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.

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

[24]  Stephen P. Jackson,et al.  A role for Saccharomyces cerevisiae histone H2A in DNA repair , 2000, Nature.

[25]  D. Toczyski,et al.  Colocalization of Mec1 and Mrc1 is sufficient for Rad53 phosphorylation in vivo , 2012, Molecular biology of the cell.

[26]  P. Plevani,et al.  Phosphorylation of the Budding Yeast 9-1-1 Complex Is Required for Dpb11 Function in the Full Activation of the UV-Induced DNA Damage Checkpoint , 2008, Molecular and Cellular Biology.

[27]  J. Diffley,et al.  Dpb11 coordinates Mec1 kinase activation with cell cycle-regulated Rad9 recruitment , 2011, The EMBO journal.

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

[29]  P. Burgers,et al.  Yeast DNA Replication Protein Dpb11 Activates the Mec1/ATR Checkpoint Kinase* , 2008, Journal of Biological Chemistry.

[30]  D. Toczyski,et al.  Colocalization of sensors is sufficient to activate the DNA damage checkpoint in the absence of damage. , 2008, Molecular cell.

[31]  D. Cortez,et al.  ATRIP Oligomerization Is Required for ATR-dependent Checkpoint Signaling* , 2005, Journal of Biological Chemistry.

[32]  G. Natsoulis,et al.  5-Fluoroorotic acid as a selective agent in yeast molecular genetics. , 1987, Methods in enzymology.

[33]  Jerzy Majka,et al.  The checkpoint clamp activates Mec1 kinase during initiation of the DNA damage checkpoint. , 2006, Molecular cell.

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

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

[36]  J. Glover,et al.  ATR autophosphorylation as a molecular switch for checkpoint activation. , 2011, Molecular cell.

[37]  W. Chazin,et al.  Function of a Conserved Checkpoint Recruitment Domain in ATRIP Proteins , 2007, Molecular and Cellular Biology.

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

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

[40]  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.

[41]  R. Zhao,et al.  TopBP1 activates ATR through ATRIP and a PIKK regulatory domain. , 2008, Genes & development.

[42]  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.

[43]  D. Durocher,et al.  Saccharomyces cerevisiae Rad9 Acts as a Mec1 Adaptor to Allow Rad53 Activation , 2005, Current Biology.

[44]  P. Burgers,et al.  The Unstructured C-terminal Tail of Yeast Dpb11 (Human TopBP1) Protein Is Dispensable for DNA Replication and the S Phase Checkpoint but Required for the G2/M Checkpoint* , 2011, The Journal of Biological Chemistry.

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

[46]  M. Kimura,et al.  Amino‐terminal domain of ATRIP contributes to intranuclear relocation of the ATR–ATRIP complex following DNA damage , 2004, FEBS letters.

[47]  I. Dobbie,et al.  Histone H2A phosphorylation and H3 methylation are required for a novel Rad9 DSB repair function following checkpoint activation. , 2006, DNA repair.

[48]  K. Cimprich,et al.  ATR: an essential regulator of genome integrity , 2008, Nature Reviews Molecular Cell Biology.

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

[50]  P. Burgers,et al.  Lagging strand maturation factor Dna2 is a component of the replication checkpoint initiation machinery. , 2013, Genes & development.

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

[52]  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.

[53]  K. Sugimoto,et al.  Requirement of the Mre11 Complex and Exonuclease 1 for Activation of the Mec1 Signaling Pathway , 2004, Molecular and Cellular Biology.

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

[55]  P. Burgers,et al.  The unstructured C-terminal tail of the 9-1-1 clamp subunit Ddc1 activates Mec1/ATR via two distinct mechanisms. , 2009, Molecular cell.

[56]  P. Sung,et al.  Activation of Protein Kinase Tel1 through Recognition of Protein-Bound DNA Ends , 2011, Molecular and Cellular Biology.

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

[58]  K. Sugimoto,et al.  Subtelomere-binding protein Tbf1 and telomere-binding protein Rap1 collaborate to inhibit localization of the Mre11 complex to DNA ends in budding yeast , 2012, Molecular biology of the cell.

[59]  E. Nam,et al.  Dpb11 activates the Mec1–Ddc2 complex , 2008, Proceedings of the National Academy of Sciences.

[60]  L. Zou,et al.  ATRIP associates with replication protein A-coated ssDNA through multiple interactions. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[61]  Haico van Attikum,et al.  The histone code at DNA breaks: a guide to repair? , 2005, Nature Reviews Molecular Cell Biology.

[62]  Sara K. Binz,et al.  Replication Protein A Directs Loading of the DNA Damage Checkpoint Clamp to 5′-DNA Junctions* , 2006, Journal of Biological Chemistry.

[63]  S. Leem,et al.  Dpb11, which interacts with DNA polymerase II(epsilon) in Saccharomyces cerevisiae, has a dual role in S-phase progression and at a cell cycle checkpoint. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[64]  Robert J. D. Reid,et al.  Cloning-free genome alterations in Saccharomyces cerevisiae using adaptamer-mediated PCR. , 2002, Methods in enzymology.