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

https://doi.org/10.1016/j.bbagen.2020.129730Get rights and content

Highlights

  • Human Exo1 is predominately an extended monomer in solution.

  • Human Exo1 C-terminal region forms a relaxed β-sheet conformation.

  • PCNA and 14–3-3 proteins interact with the C-terminal region of hExo1 to regulate its resection activity.

  • 14–3-3 proteins exhibit positive cooperative inhibition on hExo1.

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.

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.

Introduction

There are a number of surveillance and repair systems that shield the human genome from insult in order to conserve its integrity and stability. The most severe lesions that these systems guard against are DNA double strand breaks (DSBs) whereby the phosphodiester backbone of both DNA strands are simultaneously cleaved. Their efficient repair is critical, with unchecked damage resulting in genomic instability, premature aging, immunodeficiency and cancer [1,2]. Genomic stability is ensured if repair occurs before replication or transcription and multiple systems have evolved, across both prokaryotes and eukaryotes, to achieve this [[3], [4], [5], [6], [7], [8], [9], [10]]. In eukaryotes, upon detection of a DSB, two major components of the DNA lesion response system are activated: DNA repair and cell cycle arrest. Repair is initiated by recognition and binding to the free DNA ends and the subsequent process of 5′-to-3’ DNA resection. This yields a single-stranded DNA (ssDNA) structure of tens or hundreds of bases in length at the position of the double strand break [9,11,12]. Next, either non-homologous end joining (NHEJ) or homologous recombination (HR) is employed to repair the DSB, the former being faster but inherently mutagenic, while the latter ensures accurate repair. DNA end resection promotes HR and hinders non-homologous end joining [7,13]. Occurrence of DSBs, induces the checkpoint response system, which coordinates the repair of the DSB with other processes of the cell cycle, for example, promoting cell senescence or cell death if the DNA is severely damaged [14,15]. The checkpoint response induction depends on activation of ATM and ATR kinases, which initiate downstream responses to the DNA damage via protein phosphorylation cascades [16,17]. DNA end resection promotes activation of ATR, but mitigates the activation of ATM [[18], [19], [20]]. Therefore, resection is a crucial factor in determining the mode of DNA damage repair response [[21], [22], [23], [24]].

Human exonuclease1 (hExo1), an 846 amino acid protein, is a member of the Rad2/XPG nuclease family. Its cellular function has been associated with many DNA metabolic processes, including DNA mismatch repair (MMR), micro-mediated end-joining, homologous recombination (HR), and replication [[25], [26], [27], [28], [29], [30]]. The nucleolytic resection activity of hExo1 is modulated by interaction partners including MutSα (MSH2/MSH6 complex) [31], PCNA [32], 14–3-3 s [33,41,60], 9–1-1 complex [34], MRN [35], SOSS1 [36], HELB [37], Shieldin [38,39], and DYNLL1 [40]. Human Exo1 was reported to consist of a nuclease domain (residues 1–507), central domain (residues 508–750) and C-terminal domain (residues 751–846) [41]. The nuclease domain promotes binding to DNA and contains multiple cysteine and glutamate residues essential for Mg2+ binding [42]. To date, most biochemical studies have focused on the N-terminal region of hExo1 [[42], [43], [44]], which is known to be well folded. The crystal structure of the first 348 amino acids of hExo1 is available [42], however the remaining 498 amino acids have not been structurally characterized.

Recently, a model for the regulation of hExo1 in DNA end resection has been proposed [41], whereby association of hExo1 with DNA damage is controlled through protein-protein interactions with PCNA and 14–3-3 proteins. PCNA interacts with the PIP-Box, spanning residues 751–846, of the C-terminal region of hExo1 to promote retention at sites of DNA damage and processive resection of DNA breaks. The 14–3-3 proteins associate with the central domain (residues 508–750) of hExo1 and hinder PCNA binding, limiting the resection activity of hExo1 [41]. The precise mechanism by which this is achieved remains elusive and in this study we employ biophysical and enzymatic assays to better understand the role of the C-terminal region in the regulation of the DNA resection activity of hExo1. These analyses suggests that the C-terminal has a highly extended structure with a tendency to adopt a novel left-handed β-sheet structure. The C-terminus may exhibit a transient fluctuating structure that could play a role in the regulation of the activity of hExo1. Interaction with 14–3-3 protein has a cooperative inhibitory effect, which, is likely to be as a result of induced conformational change in the C-terminus of hExo1 that brings about allosteric transition upon binding partner proteins.

Section snippets

Expression of recombinant proteins

Propagated plasmid (pFastbacI-hExo1b) was isolated from TOP10 chemically competent E. coli (Invitrogen) using PureLink™ HiPure Plasmid DNA Miniprep Kit (Invitrogen). Purified plasmid DNA was used to transform competent DH10Bac E.coli cells that contain a baculovirus shuttle vector and a helper plasmid to generate recombinant bacmid DNA. After PCR verification that the genes had been inserted, the bacmids were used to transfect Sf21 cells using Cellfectin II transfection reagent (Invitrogen),

Recombinant proteins purification

Recombinant proteins were purified to near homogeneity. SDS-PAGE analysis of the purified proteins showed that all the samples analysed were highly pure and suffered no proteolytic degradation (Fig. 1A). With the full length protein offering an opportunity to biophysically characterise the C-terminal region of the protein for the first time. Similarly, the elution profile for gel filtration elution of hExo1 using a Superose 12 10/300 GL column (GE Healthcare) shows that hExo1 behaved as a

Discussion

To date, most biochemical studies of hExo1 have focused on the amino terminal domain of the full-length protein [[42], [43], [44]]. The crystal structure of the first 348 amino acids is available, however, the remaining 498 amino acids have not yet been characterized structurally. This study has uncovered that hExo1 consist of a folded N-terminal nuclease domain (amino acid residues 1–350) and a highly extended C-terminal region (amino acid residues 351–846) which is known to interact with

Credit author statement

The research was conceived by Aminu Argungu Umar, Mark Odell and David J. Scott. Susan Liddell, Karishma Asiani and Stephen Carr helped with protein expression studies. Rohanah Hussain and Giuliano Siligardi aided in acquisition and interpretation of circular dichroism and DLS data. Darren M. Gowers aided in interpretation of enzymatic assays. Gemma Harris assisted with the analytical ultracentrifugation experiments.

Proteins used in this study

NCBI codes for proteins: hExo1 (AAD13754), 14–3-3 zeta (NP_663723), 14–3-3 epsilon (NP_006752) and PCNA (CAG38740).

Funding

This project was funded by the Nigerian government through Tertiary Education Trust Fund (TetFund). Circular Dichroism beamtime on B23 of the Diamond Light Source was funded through a beam time award to DJS (SM15313–1/SM14658–1/SM14430–1). DJS is a Senior Neutron and Molecular Biology Fellow sponsored by the Science and Technology Facilities Council (UK). This work was carried out, in part, at the Research Complex at Harwell, Oxfordshire, UK.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

We thank Paul Modrich and Lene Juel Rasmussen for human Exo1 and human 14-3-3 expression constructs, respectively.

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