Department of Microbiology & Immunology, Columbia University Irving Medical Center, New York, NY 10032, USA.
Department of Genetics & Development, Columbia University Irving Medical Center, New York, NY 10032, USA.
Nucleic Acids Res. 2019 Dec 2;47(21):11238-11249. doi: 10.1093/nar/gkz814.
Sae2 functions in the DNA damage response by controlling Mre11-Rad50-Xrs2 (MRX)-catalyzed end resection, an essential step for homology-dependent repair of double-strand breaks (DSBs), and by attenuating DNA damage checkpoint signaling. Phosphorylation of Sae2 by cyclin-dependent kinase (CDK1/Cdc28) activates the Mre11 endonuclease, while the physiological role of Sae2 phosphorylation by Mec1 and Tel1 checkpoint kinases is not fully understood. Here, we compare the phenotype of sae2 mutants lacking the main CDK (sae2-S267A) or Mec1 and Tel1 phosphorylation sites (sae2-5A) with sae2Δ and Mre11 nuclease defective (mre11-nd) mutants. The phosphorylation-site mutations confer DNA damage sensitivity, but not to the same extent as sae2Δ. The sae2-S267A mutation is epistatic to mre11-nd for camptothecin (CPT) sensitivity and synergizes with sgs1Δ, whereas sae2-5A synergizes with mre11-nd and exhibits epistasis with sgs1Δ. We find that attenuation of checkpoint signaling by Sae2 is mostly independent of Mre11 endonuclease activation but requires Mec1 and Tel1-dependent phosphorylation of Sae2. These results support a model whereby CDK-catalyzed phosphorylation of Sae2 activates resection via Mre11 endonuclease, whereas Sae2 phosphorylation by Mec1 and Tel1 promotes resection by the Dna2-Sgs1 and Exo1 pathways indirectly by dampening the DNA damage response.
Sae2 通过控制 Mre11-Rad50-Xrs2 (MRX) 催化的末端切除在 DNA 损伤反应中发挥作用，末端切除是同源依赖性双链断裂 (DSB) 修复的必要步骤，并且减弱了 DNA 损伤检查点信号。细胞周期蛋白依赖性激酶 (CDK1/Cdc28) 对 Sae2 的磷酸化激活了 Mre11 内切酶，而 Mec1 和 Tel1 检查点激酶对 Sae2 的磷酸化的生理作用尚未完全阐明。在这里，我们比较了缺乏主要 CDK (sae2-S267A) 或 Mec1 和 Tel1 磷酸化位点 (sae2-5A) 的 sae2 突变体与 sae2Δ 和 Mre11 核酸酶缺陷 (mre11-nd) 突变体的表型。磷酸化位点突变赋予了 DNA 损伤敏感性，但不如 sae2Δ 那么显著。sae2-S267A 突变对喜树碱 (CPT) 敏感性与 mre11-nd 呈上位性，并且与 sgs1Δ 协同作用，而 sae2-5A 与 mre11-nd 协同作用，并与 sgs1Δ 呈上位性。我们发现，Sae2 检查点信号的衰减在很大程度上独立于 Mre11 内切酶的激活，但需要 Mec1 和 Tel1 依赖性的 Sae2 磷酸化。这些结果支持了这样一种模型，即 CDK 催化的 Sae2 磷酸化通过 Mre11 内切酶激活切除，而 Mec1 和 Tel1 对 Sae2 的磷酸化通过间接抑制 DNA 损伤反应来促进 Dna2-Sgs1 和 Exo1 途径的切除。
