Role of intrinsic disorder in human exonuclease1 regulation

Human exonuclease1 (hExo1) is a member of the eukaryotic nuclease family that includes Rad2/Xeroderma pigmentosum complementation group G (XPG), flap endonuclease1 (FEN1) and gap endonuclease1 (GEN1). Human exonuclease1 is involved in multiple DNA metabolism processes, including DNA repair and replication. Most of the fundamental roles of Exo1 have been described in yeast. In this study, hExo1 protein was over expressed from both insect cells and bacteria and over expressed protein was purified to near homogeneity. In this research project, a biochemical characterization of full-length hExo1 is reported. As well as assaying hExo1 on different dsDNA substrates, the factors essential for the thermodynamic stability of hExo1 were determined. It is shown in this study that resection activity and stability of hExo1 on dsDNA is modulated by temperature, pH and salt concentration. The DNA end resection process is a guiding principle to cellular response during DNA double strand break lesion and is pivotal for genome maintenance. Even though insufficient DNA resection restrains homology-directed repair mechanisms and the activation of ATR (ataxia telangiectasia and Rad3 related)-dependent checkpoint, over-resection results in production of an excessive single-stranded DNA that could lead to genomic instability. Nonetheless, the control mechanisms for DNA end resection are not yet understood fully. In this study it is shown that the major resection nuclease hExo1 is both positively and negatively controlled by protein-protein interactions to enable a proper DNA end resection mechanism. This report show that 14-3-3ζ 14-3-3ε proteins interact with the C-terminus region of hExo1 and allosterically control hExo1 DNA end-resection activity while PCNA sliding clamp increases the DNA end resection activity by hExo1. Circular dichroism shows that the C-terminus region of hExo1 is intrinsically disordered with significant polyproline type II conformations. Dynamic Light Scattering and Sedimentation Velocity Analytical Ultracentrifugation results show that a monomeric, partly intrinsically disordered, form persists for hExo1 in solution with an expanded hydrodynamic radius of 118 Å. Taking into consideration the structural propensity of hExo1 and the fact that, more often the binding sites for the 14-3-3 proteins are found within the unstructured regions, this study propose that the disorder-to-order transition of the ligand molecule’s structure might be a model of how hExo1 is negatively control by the 14-3-3 proteins. Results of this project work provide crucial insights into a pioneering process of DNA end resection regulation a critical event in genome maintenance and may be implicative in cancer treatment

Production and Screening of Streptomyces-Extracellular Chitinase

The aim of this research was to produce Streptomyces-extracellular chitinase and screen its antifungal activity on a clinically isolated Candida albicans. The Streptomyces were isolated from an agricultural farmland; they were identified and screened for the chitinase production. Effects of time, temperature, pH and nitrogen sources on the chitinase production were determined using standard methods. Ammonium sulphate precipitation was used to partially purify the chitinase. Protein concentrations were determined spectrophotometrically using bovine serum albumin as standard. Agar-well diffusion method was used to evaluate the antifungal activity of the chitinase on C. albicans. The isolated Streptomyces were of three (3) strains, and all the strains are Gram positive, catalase positive, oxidase positive while, Strain A and C are indole positive and only Strain B is citrate positive. The maximum chitinase production was at 72 h, 40°C and when yeast extract was used as the nitrogen source. Ammonium sulphate (80%) precipitation yielded the highest enzyme activity of 39.0U/ml. The maximum enzyme activity was observed at temperature of 40oC, pH 5.5 and 1.0% colloidal chitin (substrate). The partially purified chitinase showed a zone of inhibition of 20.11 ± 1.26 mm against the Candida albicans. This result has no significant difference (P>0.05) when compared with that of the standard drug (Fluconazole) with 21.42 ± 0.08 mm zone of inhibition. These findings suggest that Streptomyces at favourable conditions produce chitinase, and this enzyme can be used as an antifungal agent on Candida albicans and other chitin containing fungi

Role of intrinsic disorder in human exonuclease1 regulation

Human exonuclease1 (hExo1) is a member of the eukaryotic nuclease family that includes Rad2/Xeroderma pigmentosum complementation group G (XPG), flap endonuclease1 (FEN1) and gap endonuclease1 (GEN1). Human exonuclease1 is involved in multiple DNA metabolism processes, including DNA repair and replication. Most of the fundamental roles of Exo1 have been described in yeast. In this study, hExo1 protein was over expressed from both insect cells and bacteria and over expressed protein was purified to near homogeneity. In this research project, a biochemical characterization of full-length hExo1 is reported. As well as assaying hExo1 on different dsDNA substrates, the factors essential for the thermodynamic stability of hExo1 were determined. It is shown in this study that resection activity and stability of hExo1 on dsDNA is modulated by temperature, pH and salt concentration. The DNA end resection process is a guiding principle to cellular response during DNA double strand break lesion and is pivotal for genome maintenance. Even though insufficient DNA resection restrains homology-directed repair mechanisms and the activation of ATR (ataxia telangiectasia and Rad3 related)-dependent checkpoint, over-resection results in production of an excessive single-stranded DNA that could lead to genomic instability. Nonetheless, the control mechanisms for DNA end resection are not yet understood fully. In this study it is shown that the major resection nuclease hExo1 is both positively and negatively controlled by protein-protein interactions to enable a proper DNA end resection mechanism. This report show that 14-3-3ζ 14-3-3ε proteins interact with the C-terminus region of hExo1 and allosterically control hExo1 DNA end-resection activity while PCNA sliding clamp increases the DNA end resection activity by hExo1. Circular dichroism shows that the C-terminus region of hExo1 is intrinsically disordered with significant polyproline type II conformations. Dynamic Light Scattering and Sedimentation Velocity Analytical Ultracentrifugation results show that a monomeric, partly intrinsically disordered, form persists for hExo1 in solution with an expanded hydrodynamic radius of 118 Å. Taking into consideration the structural propensity of hExo1 and the fact that, more often the binding sites for the 14-3-3 proteins are found within the unstructured regions, this study propose that the disorder-to-order transition of the ligand molecule’s structure might be a model of how hExo1 is negatively control by the 14-3-3 proteins. Results of this project work provide crucial insights into a pioneering process of DNA end resection regulation a critical event in genome maintenance and may be implicative in cancer treatment

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

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