It is hoped that the inhibition of the pathways that replicate and repair DNA will facilitate the development of new therapies in the fields of cancer treatment and antibiotics. For example, DNA ligases function in both DNA replication and repair pathways, sealing single-strand breaks in duplex DNA. The difference in cosubstrate specificity between the essential DNA ligases in bacteria and eukaryotes makes these enzymes potential drug targets for a novel class of antibiotics. Traditionally, DNA ligase activities have been measured using electrophoretic separation of DNA fragments to detect changes in the length of a reporter-labelled strand. However, this assay is slow and laborious with limited capacity. To allow screening of potential inhibitors and more extensive research in the field of DNA repair and replication, a new assay is required that has both higher-throughput and versatility in the experiments that can be performed. This thesis presents the development of such an assay using DNA hairpins labelled at one terminus by biotin and at the other by a fluorophore and immobilised in streptavidin coated microtiter plates through the biotin-streptavidin interaction. Loss or retention of fluorescence following denaturation of the hairpin reports on the integrity of the DNA backbone, which is altered by DNA ligase or nuclease activity. Using this immobilised DNA hairpin assay, the activities of the NAD+-dependent DNA ligases from Escherichia coli, Streptomyces coelicolor and Mycobacterium tuberculosis were studied. DNA ligase from bacteriophage T4 was also studied as a representative enzyme from the ATP-dependent class of DNA ligases. For the study of nuclease activity, restriction enzymes Alul, Ddel, KpnI, and Rsal were used, and the nicking enzyme N. BbvCI A. The activities of N. BbvCI A and DNA ligase from E. coli were combined to demonstrate sequential activity on a single sample of immobilised DNA hairpins. Such experiments provide a basis for reconstituting complete reaction pathways (such as DNA base excision repair) on single samples of immobilised substrate. The immobilised DNA hairpin assay was further employed tostudy the mixed activities of HeLa nuclear and whole cell extracts, and cell extracts from E. coli BL21 pLysS. Using the developed assay, the difference in activity between E. coli DNA ligase and the L15F variant is shown to be due to a much slower rate of enzyme adenylation in the latter. Finally, a theoretical framework to elucidate kinetic and mechanistic aspects of the immobilised DNA hairpin assay is presented. Thus, an assay is presented that can detect the activity of a wide range of DNA metabolizing enzymes in a format compatible with high throughput analyses. The assay is suitable for detecting activity both from purified enzymes and crude cell extracts. By exploiting the heterogeneous nature of the assay, novel experiments are possible that greatly facilitate analysis of DNA processing pathways when compared to the traditional methods.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:439938 |
| Date | January 2007 |
| Creators | Scott, Benjamin Oliver Seager |
| Publisher | University of East Anglia |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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