Kaur, Loveleen (2009) Developing as assay to screen inhibitors for various ATP-dependent ligases. Doctoral thesis, University of Westminster.
DNA ligases (EC.22.214.171.124) are key enzymes that catalyze the formation of phosphodiester bonds at single-stranded or double-stranded breaks between adjacent 5’-PO4 and 3’-OH groups of DNA. These enzymes are essential guardians of genomic integrity and have recently been drawing a lot of attention as novel therapeutic targets in anti-bacterial and anticancer therapies. A novel, non-electrophoretic assay method, based on the strength of interaction of the oligonucleotides with Q-sepharose (a strong anion exchanger), was developed to screen inhibitors of DNA ligases from natural product pools as well as chemical libraries. The binding affinities to Q-sepharose resin of a nicked DNA substrate (created from a 30-mer hairpin oligonucleotide and complementary 32P-labelled 6-mer oligonucleotide) and its sealed, ligated product (36-mer) were determined. Initial optimisation studies were performed with T4 DNA ligase, PBCV-1 DNA ligase and a catalytically active form of human DNA ligase I in the presence of doxorubicin (inhibitor of ATP-dependent ligases). These results when analysed in parallel between the conventional electrophoretic assay and the labelled nick-sealing assay showed that the newly developed assay is a reliable non-electrophoretic method in identifying potent DNA ligase inhibitors. The feasibility of the assay was tested in screening a collection of whole cell mass extracts, obtained from a natural product library from Basidiomycetes, in 96-well format. A novel single DNA ligase was identified, expressed and characterised from Trichomonas vaginalis (TV), a pathogenic protozoan parasite. Protein sequence analysis of TV DNA ligase indicates a strong sequence similarity to DNA ligase I homologues. The activity of recombinant TV DNA ligase I (TVlig) was investigated using protein expressed in E.coli cells. The TVlig gene product is 76 kDa and showed optimal ligation activity on a nicked DNA substrate at pH 7-8 in the presence of 1 mM ATP and (8- 20) mM MgCl2 at 30-38oC. The inhibition of the only DNA ligase present in T. vaginalis might suggest for a rational approach to stop replication and hence propagation of the parasite during infection.
|Item Type:||Thesis (Doctoral)|
|Subjects:||University of Westminster > Science and Technology > Life Sciences, School of (No longer in use)|
|Depositing User:||Miss Nina Watts|
|Date Deposited:||17 Aug 2010 09:08|
|Last Modified:||17 Aug 2010 09:08|
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