Department of Science, Systems and Models, Roskilde University, Denmark.
DNA Repair (Amst). 2012 Mar 1;11(3):267-77. doi: 10.1016/j.dnarep.2011.11.007. Epub 2012 Jan 4.
Human exonuclease 1 (hEXO1) acts directly in diverse DNA processing events, including replication, mismatch repair (MMR), and double strand break repair (DSBR), and it was also recently described to function as damage sensor and apoptosis inducer following DNA damage. In contrast, 14-3-3 proteins are regulatory phosphorserine/threonine binding proteins involved in the control of diverse cellular events, including cell cycle checkpoint and apoptosis signaling. hEXO1 is regulated by post-translation Ser/Thr phosphorylation in a yet not fully clarified manner, but evidently three phosphorylation sites are specifically induced by replication inhibition leading to protein ubiquitination and degradation. We demonstrate direct and robust interaction between hEXO1 and six of the seven 14-3-3 isoforms in vitro, suggestive of a novel protein interaction network between DNA repair and cell cycle control. Binding experiments reveal weak affinity of the more selective isoform 14-3-3σ but both 14-3-3 isoforms η and σ significantly stimulate hEXO1 activity, indicating that these regulatory proteins exert a common regulation mode on hEXO1. Results demonstrate that binding involves the phosphorable amino acid S746 in hEXO1 and most likely a second unidentified binding motif. 14-3-3 associations do not appear to directly influence hEXO1 in vitro nuclease activity or in vitro DNA replication initiation. Moreover, specific phosphorylation variants, including hEXO1 S746A, are efficiently imported to the nucleus; to associate with PCNA in distinct replication foci and respond to DNA double strand breaks (DSBs), indicating that 14-3-3 binding does not involve regulating the subcellular distribution of hEXO1. Altogether, these results suggest that association may be related to regulation of hEXO1 availability during the DNA damage response to plausibly prevent extensive DNA resection at the damage site, as supported by recent studies.
人源核酸外切酶 1（hEXO1）直接参与多种 DNA 加工事件，包括复制、错配修复（MMR）和双链断裂修复（DSBR），最近还被描述为 DNA 损伤后作为损伤传感器和凋亡诱导因子发挥作用。相比之下，14-3-3 蛋白是调节丝氨酸/苏氨酸结合蛋白，参与多种细胞事件的控制，包括细胞周期检查点和凋亡信号转导。hEXO1 的翻译后 Ser/Thr 磷酸化调节方式尚未完全阐明，但显然有三个磷酸化位点被复制抑制特异性诱导，导致蛋白质泛素化和降解。我们在体外证明了 hEXO1 与七种 14-3-3 同工型中的六种的直接和强相互作用，提示 DNA 修复和细胞周期控制之间存在新的蛋白质相互作用网络。结合实验揭示了更具选择性的同工型 14-3-3σ的弱亲和力，但 14-3-3 同工型 η和σ都显著刺激 hEXO1 的活性，表明这些调节蛋白对 hEXO1 具有共同的调节模式。结果表明，结合涉及 hEXO1 中的可磷酸化氨基酸 S746，很可能还有第二个未识别的结合基序。14-3-3 结合似乎不会直接影响 hEXO1 的体外核酸酶活性或体外 DNA 复制起始。此外，包括 hEXO1 S746A 在内的特定磷酸化变体能够有效地被导入细胞核；与 PCNA 形成不同的复制焦点，并对 DNA 双链断裂（DSBs）作出反应，表明 14-3-3 结合不涉及调节 hEXO1 的亚细胞分布。总之，这些结果表明，这种结合可能与 hEXO1 在 DNA 损伤反应期间的可用性调节有关，可能是为了防止在损伤部位进行广泛的 DNA 切除，这与最近的研究结果一致。
