Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO 63110, USA.
Mol Cell. 2009 Dec 11;36(5):743-53. doi: 10.1016/j.molcel.2009.10.014.
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.
DNA 损伤检查点途径通过响应 DNA 损伤和复制应激来防止细胞周期的进行。在酿酒酵母中，Mec1-Ddc2（人类 ATR-ATRIP）是主要的检查点蛋白激酶。生化研究已经确定了两个因素，即 9-1-1 检查点夹和 Dpb11/TopBP1 复制蛋白，作为 Mec1/ATR 的潜在激活因子。在这里，我们表明，9-1-1 的 Ddc1 亚基实现了 G1 期检查点对 Mec1 的激活，而 Dpb11 则是可有可无的。然而，在 G2 期，9-1-1 通过两种不同的机制激活 Mec1。一种机制涉及 Ddc1 对 Mec1 的直接激活，而第二种机制则通过 Ddc1 T602 磷酸化介导的 Dpb11 募集进行。位于 Ddc1 的两个广泛分离的保守模体中的两个芳香族残基 W352 和 W544 对于体外 Mec1 的激活和 G1 中的检查点功能至关重要。值得注意的是，融合了两个含色氨酸的模体的小肽能够有效地激活 Mec1。
