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.
Following genotoxic insults, eukaryotic cells trigger a signal transduction cascade known as the DNA damage checkpoint response, which involves the loading onto DNA of an apical kinase and several downstream factors. Chromatin modifications play an important role in recruiting checkpoint proteins. In budding yeast, methylated H3-K79 is bound by the checkpoint factor Rad9. Loss of Dot1 prevents H3-K79 methylation, leading to a checkpoint defect in the G(1) phase of the cell cycle and to a reduction of checkpoint activation in mitosis, suggesting that another pathway contributes to Rad9 recruitment in M phase. We found that the replication factor Dpb11 is the keystone of this second pathway. dot1Delta dpb11-1 mutant cells are sensitive to UV or Zeocin treatment and cannot activate Rad53 if irradiated in M phase. Our data suggest that Dpb11 is held in proximity to damaged DNA through an interaction with the phosphorylated 9-1-1 complex, leading to Mec1-dependent phosphorylation of Rad9. Dpb11 is also phosphorylated after DNA damage, and this modification is lost in a nonphosphorylatable ddc1-T602A mutant. Finally, we show that, in vivo, Dpb11 cooperates with Dot1 in promoting Rad9 phosphorylation but also contributes to the full activation of Mec1 kinase.
Dipartimento di Scienze Biomolecolari e Biotecnologie, Università degli Studi di Milano, Via Celoria 26, 20133 Milan, Italy.
Fabio
F
Puddu
Magda
M
Granata
Lisa
L
Di Nola
Alessia
A
Balestrini
Gabriele
G
Piergiovanni
Federico
F
Lazzaro
Michele
M
Giannattasio
Paolo
P
Plevani
Marco
M
Muzi-Falconi
D018797:Cell Cycle Proteins / Q000378:metabolism*; D016384:Consensus Sequence; D053903:DNA Breaks, Double-Stranded / Q000528:radiation effects; D004249:DNA Damage; D004789:Enzyme Activation / Q000528:radiation effects; D046912:Multiprotein Complexes / Q000378:metabolism*; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D004718:Saccharomycetales / Q000166:cytology / Q000201:enzymology / Q000378:metabolism* / Q000528:radiation effects*; D014466:Ultraviolet Rays
D018797:Cell Cycle Proteins; C097185:DPB11 protein, S cerevisiae; D046912:Multiprotein Complexes; D029701:Saccharomyces cerevisiae Proteins; D019000:Phosphotyrosine