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

DOI: 10.1016/j.bbagen.2020.129730

Authors: Aminu Argungu Umar (University of Nottingham; Kebbi State University of Science and Technology) , Susan Liddell (University of Nottingham) , Rohanah Hussain (Diamond Light Source) , Giuliano Siligardi (Diamond Light Source) , Gemma Harris (Research Complex at Harwell) , Stephen Carr (Research Complex at Harwell) , Karishma Asiani (University of Nottingham) , Darren M. Gowers (University of Portsmouth) , Mark Odell (University of Westminster) , David J. Scott (University of Nottingham; Research Complex at Harwell; ISIS Spallation Neutron and Muon Source)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Biochimica Et Biophysica Acta (bba) - General Subjects , VOL 1864

State: Published (Approved)
Published: December 2020
Diamond Proposal Number(s): 15313 , 14658 , 14430

Abstract: 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.

Journal Keywords: Intrinsic disorder; Circular dichroism; Allostery; Exonuclease; Enzyme assay

Subject Areas: Biology and Bio-materials, Chemistry


Instruments: B23-Circular Dichroism