TY - JOUR
T1 - The Unstructured C-Terminal Tail of the 9-1-1 Clamp Subunit Ddc1 Activates Mec1/ATR via Two Distinct Mechanisms
AU - Navadgi-Patil, Vasundhara M.
AU - Burgers, Peter M.
N1 - Funding Information:
We thank Jurek Majka for help and advice during the initial stages of this work, John Majors for critical discussions, and Carrie Stith for expert technical assistance. This work was supported in part by NIH grants GM32431 and GM083970.
PY - 2009/12/11
Y1 - 2009/12/11
N2 - DNA damage checkpoint pathways operate to prevent cell-cycle progression in response to DNA damage and replication stress. In S. cerevisiae, Mec1-Ddc2 (human ATR-ATRIP) is the principal checkpoint protein kinase. Biochemical studies have identified two factors, the 9-1-1 checkpoint clamp and the Dpb11/TopBP1 replication protein, as potential activators of Mec1/ATR. Here, we show that G1 phase checkpoint activation of Mec1 is achieved by the Ddc1 subunit of 9-1-1, while Dpb11 is dispensable. However, in G2, 9-1-1 activates Mec1 by two distinct mechanisms. One mechanism involves direct activation of Mec1 by Ddc1, while the second proceeds by Dpb11 recruitment mediated through Ddc1 T602 phosphorylation. Two aromatic residues, W352 and W544, localized to two widely separated, conserved motifs of Ddc1, are essential for Mec1 activation in vitro and checkpoint function in G1. Remarkably, small peptides that fuse the two tryptophan-containing motifs together are proficient in activating Mec1.
AB - DNA damage checkpoint pathways operate to prevent cell-cycle progression in response to DNA damage and replication stress. In S. cerevisiae, Mec1-Ddc2 (human ATR-ATRIP) is the principal checkpoint protein kinase. Biochemical studies have identified two factors, the 9-1-1 checkpoint clamp and the Dpb11/TopBP1 replication protein, as potential activators of Mec1/ATR. Here, we show that G1 phase checkpoint activation of Mec1 is achieved by the Ddc1 subunit of 9-1-1, while Dpb11 is dispensable. However, in G2, 9-1-1 activates Mec1 by two distinct mechanisms. One mechanism involves direct activation of Mec1 by Ddc1, while the second proceeds by Dpb11 recruitment mediated through Ddc1 T602 phosphorylation. Two aromatic residues, W352 and W544, localized to two widely separated, conserved motifs of Ddc1, are essential for Mec1 activation in vitro and checkpoint function in G1. Remarkably, small peptides that fuse the two tryptophan-containing motifs together are proficient in activating Mec1.
KW - DNA
KW - PROTEINS
UR - http://www.scopus.com/inward/record.url?scp=71149093704&partnerID=8YFLogxK
U2 - 10.1016/j.molcel.2009.10.014
DO - 10.1016/j.molcel.2009.10.014
M3 - Article
C2 - 20005839
AN - SCOPUS:71149093704
SN - 1097-2765
VL - 36
SP - 743
EP - 753
JO - Molecular cell
JF - Molecular cell
IS - 5
ER -