Wanrooij Paulina H, Tannous Elias, Kumar Sandeep, Navadgi-Patil Vasundhara M, Burgers Peter M
From the Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110, Department of Medical Biochemistry and Biophysics, Umeå University, 901 87 Umeå, Sweden, and.
From the Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110.
J Biol Chem. 2016 Jan 1;291(1):393-401. doi: 10.1074/jbc.M115.687145. Epub 2015 Oct 23.
Yeast Mec1, the ortholog of human ATR, is the apical protein kinase that initiates the cell cycle checkpoint in response to DNA damage and replication stress. The basal activity of Mec1 kinase is activated by cell cycle phase-specific activators. Three distinct activators stimulate Mec1 kinase using an intrinsically disordered domain of the protein. These are the Ddc1 subunit of the 9-1-1 checkpoint clamp (ortholog of human and Schizosaccharomyces pombe Rad9), the replication initiator Dpb11 (ortholog of human TopBP1 and S. pombe Cut5), and the multifunctional nuclease/helicase Dna2. Here, we use small peptides to determine the requirements for Mec1 activation. For Ddc1, we identify two essential aromatic amino acids in a hydrophobic environment that when fused together are proficient activators. Using this increased insight, we have been able to identify homologous motifs in S. pombe Rad9 that can activate Mec1. Furthermore, we show that a 9-amino acid Dna2-based peptide is sufficient for Mec1 activation. Studies with mutant activators suggest that binding of an activator to Mec1 is a two-step process, the first step involving the obligatory binding of essential aromatic amino acids to Mec1, followed by an enhancement in binding energy through interactions with neighboring sequences.
酵母中的Mec1是人类ATR的直系同源物，是一种顶端蛋白激酶，可在DNA损伤和复制应激时启动细胞周期检查点。Mec1激酶的基础活性由细胞周期阶段特异性激活剂激活。三种不同的激活剂利用该蛋白的一个内在无序结构域刺激Mec1激酶。这些激活剂分别是9-1-1检查点钳的Ddc1亚基（人类和粟酒裂殖酵母Rad9的直系同源物）、复制起始因子Dpb11（人类TopBP1和粟酒裂殖酵母Cut5的直系同源物）以及多功能核酸酶/解旋酶Dna2。在此，我们使用小肽来确定Mec1激活的条件。对于Ddc1，我们在一个疏水环境中鉴定出两个必需的芳香族氨基酸，将它们融合在一起时就是有效的激活剂。基于这一深入认识，我们得以在粟酒裂殖酵母Rad9中鉴定出可激活Mec1的同源基序。此外，我们表明一种基于Dna2的9氨基酸肽足以激活Mec1。对突变激活剂的研究表明，激活剂与Mec1的结合是一个两步过程，第一步涉及必需芳香族氨基酸与Mec1的强制结合，随后通过与相邻序列的相互作用增强结合能。
