title: Ddc2 mediates Mec1 activation through a Ddc1- or Dpb11-independent mechanism.
abstract: 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.
authors.affiliation: Department of Microbiology and Molecular Genetics, New Jersey Medical School, Rutgers, The State University of New Jersey, Newark, New Jersey, United States of America.
authors.forename: Amitava
authors.initials: A
authors.lastname: Bandhu
authors.affiliation: Department of Microbiology and Molecular Genetics, New Jersey Medical School, Rutgers, The State University of New Jersey, Newark, New Jersey, United States of America.
authors.forename: John
authors.initials: J
authors.lastname: Kang
authors.affiliation: Department of Microbiology and Molecular Genetics, New Jersey Medical School, Rutgers, The State University of New Jersey, Newark, New Jersey, United States of America.
authors.forename: Kenzo
authors.initials: K
authors.lastname: Fukunaga
authors.affiliation: Department of Microbiology and Molecular Genetics, New Jersey Medical School, Rutgers, The State University of New Jersey, Newark, New Jersey, United States of America.
authors.forename: Greicy
authors.initials: G
authors.lastname: Goto
authors.affiliation: Department of Microbiology and Molecular Genetics, New Jersey Medical School, Rutgers, The State University of New Jersey, Newark, New Jersey, United States of America.
authors.forename: Katsunori
authors.initials: K
authors.lastname: Sugimoto
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism*; D059447:Cell Cycle Checkpoints / Q000235:genetics*; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D004249:DNA Damage / Q000235:genetics; D006657:Histones / Q000235:genetics / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D018797:Cell Cycle Proteins; C097185:DPB11 protein, S cerevisiae; C440013:Ddc1 protein, S cerevisiae; D006657:Histones; D047908:Intracellular Signaling Peptides and Proteins; C440014:LCD1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C088937:MEC1 protein, S cerevisiae; D017346:Protein Serine-Threonine Kinases