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Allosteric inhibition of human exonuclease1 (hExo1) through a novel extended β-sheet conformation.
Biochimica et Biophysica Acta (BBA) - General Subjects ( IF 3 ) Pub Date : 2020-09-12 , DOI: 10.1016/j.bbagen.2020.129730
Aminu Argungu Umar 1 , Susan Liddell 2 , Rohanah Hussain 3 , Giuliano Siligardi 3 , Gemma Harris 4 , Stephen Carr 4 , Karishma Asiani 2 , Darren M Gowers 5 , Mark Odell 6 , David J Scott 7
Affiliation  

Background

Human Exonuclease1 (hExo1) participates in the resection of DNA double-strand breaks by generating long 3′-single-stranded DNA overhangs, critical for homology-based DNA repair and activation of the ATR-dependent checkpoint. The C-terminal region is essential for modulating the activity of hExo1, containing numerous sites of post-translational modification and binding sites for partner proteins.

Methods

Analytical Ultracentrifugation (AUC), Dynamic Light Scattering (DLS), Circular Dichroism (CD) spectroscopy and enzymatic assays.

Results

AUC and DLS indicates the C-terminal region has a highly extended structure while CD suggest a tendency to adopt a novel left-handed β-sheet structure, together implying the C-terminus may exhibit a transient fluctuating structure that could play a role in binding partner proteins known to regulate the activity of hExo1. Interaction with 14–3-3 protein has a cooperative inhibitory effect upon DNA resection activity, which indicates an allosteric transition occurs upon binding partner proteins.

Conclusions

This study has uncovered that hExo1 consist of a folded N-terminal nuclease domain and a highly extended C-terminal region which is known to interact with partner proteins that regulates the activity of hExo1. A positively cooperative mechanism of binding allows for stringent control of hExo1 activity. Such a transition would coordinate the control of hExo1 by hExo1 regulators and hence allow careful coordination of the process of DNA end resection.

Significance

The assays presented herein could be readily adapted to rapidly identify and characterise the effects of modulators of the interaction between the 14–3-3 proteins and hExo1. It is conceivable that small molecule modulators of 14–3-3 s-hExo1 interaction may serve as effective chemosensitizers for cancer therapy.



中文翻译:

通过新型扩展的β-折叠构象对人外切核酸酶1(hExo1)的变构抑制作用。

背景

人类核酸外切酶1(hExo1)通过产生长的3'-单链DNA突出端,参与DNA双链断裂的切除,这对于基于同源性的DNA修复和ATR依赖性检查点的激活至关重要。C端区域对于调节hExo1的活性至关重要,该区域包含许多翻译后修饰位点和伴侣蛋白的结合位点。

方法

分析超速离心(AUC),动态光散射(DLS),圆二色谱(CD)光谱和酶法测定。

结果

AUC和DLS表示C端区域具有高度延伸的结构,而CD则表明倾向于采用新颖的左手β-sheet结构,这意味着C端可能表现出短暂的起伏结构,可能在结合中发挥作用已知调节hExo1活性的伴侣蛋白。与14–3-3蛋白的相互作用对DNA切除活性具有协同抑制作用,这表明结合伴侣蛋白时发生了变构过渡。

结论

这项研究发现,hExo1由一个折叠的N端核酸酶结构域和一个高度延伸的C端区域组成,已知该区域会与调节hExo1活性的伴侣蛋白相互作用。结合的正向合作机制允许严格控制hExo1活性。这样的转变将协调hExo1调节剂对hExo1的控制,因此可以仔细协调DNA末端切除的过程。

意义

本文介绍的测定方法可轻松应用于快速鉴定和表征14-3-3蛋白与hExo1之间相互作用的调节剂的作用。可以想象,14–3-3 s-hExo1相互作用的小分子调节剂可作为有效的化学增敏剂用于癌症治疗。

更新日期:2020-09-16
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