Isolation and characterisation of nucleases from various biological sources for further application in nucleic acid analysis

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Isolation and characterisation of nucleases from various biological sources for further application in nucleic acid analysis

 

Author: Cheung, Tsz Shan
Title: Isolation and characterisation of nucleases from various biological sources for further application in nucleic acid analysis
Degree: Ph.D.
Year: 2013
Subject: Nucleases.
Nucleic acids -- Analysis.
Hong Kong Polytechnic University -- Dissertations
Department: Dept. of Health Technology and Informatics
Pages: xxiv, 342 p. : ill. (some col.) ; 30 cm.
Language: English
InnoPac Record: http://library.polyu.edu.hk/record=b2639090
URI: http://theses.lib.polyu.edu.hk/handle/200/7149
Abstract: Nucleases are widely used in various areas including the study of nucleic acids biochemistry, therapeutics and industrial applications. They are used in detecting polymorphisms and disease-causing mutations in nucleic acid sequences, and also used as antiviral agents or other therapeutics. More than 30 different single-strand-specific nucleases have been isolated from various sources including bacteria, fungi, plants and animals. However, only a few of them are suitable for use in research and diagnostic work. In addition, little sequence information is known about many plant enzymes that possess nuclease I characteristics. In particular, the biological role of this kind of nucleases is unclear and there are very few studies reporting the efficiencies of DNA cleavage by such nucleases. In this study, a novel single-strand-specific nuclease was isolated and functionally characterised. Eleven different plants were screened in this study. Seven out of eleven samples were found to possess the target nuclease gene that was homologous to S1 nuclease. Four novel nuclease sequences were identified from Radermachera sinica, Brassica chinesis, Brassica parachinesis and Apium graveolens var.dulce. Sequence identities, homology studies and domain/motif predictions of these sequences were characterised and determined in this study. A full-length complementary DNA (882 bp) was isolated from a potted plant, Radermachera sinica (China doll), and its protein product named as CDENDO1. Recombinant CDENDO1 protein (277 amino acid residues) was successfully cloned and purified in E. coli, and functionally characterised in this study. Recombinant CDENDO1 protein carried the S1/P1 nuclease domain characteristic of endonuclease activity and nucleic acid binding, and was classified as a member of the S1/P1 nuclease family. Recombinant CDENDO1 protein was found to behave as a single-strand-specific nuclease that was capable of hydrolysing both of single-stranded RNA and double-stranded DNA endonucleolytically. Its activity was triggered by both Ca²⁺/Mg²⁺ and inhibited by Zn²⁺/Mn²⁺. Recombinant CDENDO1 nuclease could sustain in reaction mixtures containing low concentration of beta-mercaptoethanol and dithiothreitol. The nuclease activity was totally inactivated by either heating at 80°C or 85°C for 20 minutes, or in the presence of 20 mM EDTA. Modifications or removal of the signal peptide of CDENDO1 nuclease did not affect the catalytic activity of CDENDO nuclease. Removal of the last twelve amino acids, i.e. G D S Q D D S L V A A T, at the C-terminal of the mature protein was found to enhance the protein expression. In this study, CDENDO1 nuclease and endonuclease V (Endo V) were first reported to be capable of cleaving the linker regions between nucleosome cores on chromatin in vitro. They might be involved in the programmed cell death pathway. Recombinant CDENDO1 nuclease were different from CEL I nuclease and S1 nuclease although they belonged to the same superfamily of S1/P1 nuclease. Although recombinant CDENDO1 nuclease failed to recognise single-nucleotide mismatch, it was capable of nicking at the linker DNA between nucleosomes on chromatin structure. This feature enables its potential application in chromatin studies.

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