Allosteric inhibition of human exonuclease1 (hExo1) through a novel extended β-sheet conformation

Umar, Aminu Argungu, Liddell, Susan, Hussain, Rohanah , Siligardi, Giuliano, Harris, Gemma, Carr, Stephen, Asiani, Karishma, Gowers, Darren M., Odell, Mark and Scott, David J. (2020) Allosteric inhibition of human exonuclease1 (hExo1) through a novel extended β-sheet conformation. BBA - Biochimica et Biophysica Acta - General Subjects, 1864 (12). pp. 1-9. ISSN 0304-4165

Full content URL: https://doi.org/10.1016/j.bbagen.2020.129730

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Allosteric inhibition of human exonuclease1 (hExo1) through a novel extended β-sheet conformation
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Abstract

Background: Human Exonuclease1 (hExo1) participates in the resection of DNA double-strand breaks by gen-erating 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 ten-dency 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.

Keywords:Intrinsic disorder, Circular dichroism, Allostery, Exonuclease, enzyme assay
Subjects:C Biological Sciences > C710 Applied Molecular Biology, Biophysics and Biochemistry
C Biological Sciences > C720 Biological Chemistry
C Biological Sciences > C790 Molecular Biology, Biophysics and Biochemistry not elsewhere classified
Divisions:College of Science > School of Life Sciences
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ID Code:43202
Deposited On:08 Dec 2020 09:49

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