Mec1p is essential for phosphorylation of the yeast DNA damage checkpoint protein Ddc1p, which physically interacts with Mec3p.
Checkpoints prevent DNA replication or nuclear division when chromosomes are damaged. The Saccharomyces cerevisiae DDC1 gene belongs to the RAD17, MEC3 and RAD24 epistasis group which, together with RAD9, is proposed to act at the beginning of the DNA damage checkpoint pathway. Ddc1p is periodically phosphorylated during unperturbed cell cycle and hyperphosphorylated in response to DNA damage. We demonstrate that Ddc1p interacts physically in vivo with Mec3p, and this interaction requires Rad17p. We also show that phosphorylation of Ddc1p depends on the key checkpoint protein Mec1p and also on Rad24p, Rad17p and Mec3p. This suggests that Mec1p might act together with the Rad24 group of proteins at an early step of the DNA damage checkpoint response. On the other hand, Ddc1p phosphorylation is independent of Rad53p and Rad9p. Moreover, while Ddc1p is required for Rad53p phosphorylation, it does not play any major role in the phosphorylation of the anaphase inhibitor Pds1p, which requires RAD9 and MEC1. We suggest that Rad9p and Ddc1p might function in separated branches of the DNA damage checkpoint pathway, playing different roles in determining Mec1p activity and/or substrate specificity.
Dipartimento di Genetica e di Biologia dei Microrganismi, Università degli Studi di Milano, Via Celoria 26, 20133 Milano, Italy.
V
V
Paciotti
G
G
Lucchini
P
P
Plevani
M P
MP
Longhese
D002453:Cell Cycle / Q000502:physiology; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D004249:DNA Damage / Q000502:physiology*; D004271:DNA, Fungal / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins; D009687:Nuclear Proteins / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D064249:Securin
D018797:Cell Cycle Proteins; D004271:DNA, Fungal; D005656:Fungal Proteins; D047908:Intracellular Signaling Peptides and Proteins; C099938:MEC3 protein, S cerevisiae; D009687:Nuclear Proteins; C578004:PDS1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D064249:Securin; C088937:MEC1 protein, S cerevisiae; D017346:Protein Serine-Threonine Kinases