Department of Pharmacology and Toxicology, Boonshoft School of Medicine, Wright State University, Dayton, Ohio, USA
Department of Pharmacology and Toxicology, Boonshoft School of Medicine, Wright State University, Dayton, Ohio, USA.
Mol Cell Biol. 2019 Sep 27;39(20). doi: 10.1128/MCB.00175-19. Print 2019 Oct 15.
In response to perturbed DNA replication, ATR (ataxia telangiectasia and Rad3-related) kinase is activated to initiate the checkpoint signaling necessary for maintaining genome integrity and cell survival. To better understand the signaling mechanism, we carried out a large-scale genetic screen in fission yeast looking for mutants with enhanced sensitivity to hydroxyurea. From a collection of ∼370 primary mutants, we found a few mutants in which Rad3 (ATR ortholog)-mediated phospho-signaling was significantly compromised. One such mutant carried an uncharacterized mutation in , a gene encoding an essential and highly conserved eukaryotic protein. Previous studies in various biological models have shown that Tel2 mainly functions in Tel2-Tti1-Tti2 (TTT) complex that regulates the steady-state levels of all phosphatidylinositol 3-kinase-like protein kinases, including ATR. We show here that although the levels of Rad3 and Rad3-mediated phospho-signaling in DNA damage checkpoint were moderately reduced in the mutant, the phospho-signaling in the DNA replication checkpoint was almost completely eliminated. In addition, the mutation caused telomere shortening. Since the interactions of Tel2 with Tti1 and Tti2 were significantly weakened by the mutation, destabilization of the TTT complex likely contributes to the observed checkpoint and telomere defects.
为了应对受损的 DNA 复制，ATR（共济失调毛细血管扩张症和 Rad3 相关）激酶被激活，以启动维持基因组完整性和细胞存活所必需的检查点信号。为了更好地理解信号机制，我们在裂殖酵母中进行了大规模的遗传筛选，寻找对羟基脲敏感性增强的突变体。从大约 370 个原始突变体中，我们发现了一些 Rad3（ATR 同源物）介导的磷酸化信号显著受损的突变体。其中一个这样的突变体携带一个未被表征的突变 ，该基因编码一个必需的和高度保守的真核蛋白。在各种生物模型中的先前研究表明，Tel2 主要在 Tel2-Tti1-Tti2（TTT）复合物中发挥作用，该复合物调节所有磷脂酰肌醇 3-激酶样蛋白激酶的稳态水平，包括 ATR。我们在这里表明，尽管在 突变体中，DNA 损伤检查点的 Rad3 和 Rad3 介导的磷酸化信号水平中度降低，但 DNA 复制检查点的磷酸化信号几乎完全消除。此外， 突变导致端粒缩短。由于 Tel2 与 Tti1 和 Tti2 的相互作用因突变而显著减弱，TTT 复合物的不稳定性可能导致观察到的检查点和端粒缺陷。
