As a structural analogue of adenine (6-aminopurine), 2-aminopurine (2AP) is a powerful fluorescent probe, when substituted in DNA in place of the natural adenine. Time-resolved fluorescence measurements of 2AP-labeled oligonucleotides, together with steady-state spectroscopy give us an in-depth view of DNA-enzyme interactions, especially the conformational dynamics in solution phase. Herein, this technique has been extended to the study of the transient unzipping of DNA bases, to investigate the structure of three-way junction (3WJ), and the role of base unzipping in the mechanism of human flap endonuclease (FEN). Seven 2AP-labelled 3WJs were investigated, each containing only one 2AP base in place of adenine. In four of the 3WJs, 2AP was placed in the long duplex region of an arm; while in the other three 3WJs, 2AP was placed near or in the branch point. Comparative time-resolved fluorescence measurements on the 3WJs and corresponding ssDNA and dsDNA controls were made to study the base dynamics, in particular the possibility of unzipping in the vicinity of the branch point. In combination with single-molecule FRET measurements and molecular dynamics simulations, the local and global structure of a DNA 3WJ in solution could be unraveled. It was found to adopt a Y-shaped, pyramidal structure, in which the bases adjacent to the branch point are unzipped, despite the full Watson-Crick complementarity of the molecule. Human flap endonuclease (hFEN) is divalent metal ion-dependent phosphodiesterase. hFEN carries out structure-specific hydrolysis of 5’ bifurcated DNA endonucleolytically. Cleavage occurs at a position one nucleotide into the downstream duplex region. Previous structural, biochemical and modeling studies suggested a double-nucleotide unzipping mechanism at single/double strand junctions for scissile phosphate placement. To confirm this mechanism, 2AP time-resolved fluorescence spectroscopy was used to investigate nucleotide unzipping in hFEN substrates. 2AP was substituted at positions +1 and -1 (relative to the scissile phosphodiester) respectively, in double flap substrates. A series of hFEN mutants including Y40A, R100A, K93A, were used in this study. In the experiments, ssDNA, dsDNA substrates, DNA substrate-enzyme complexes were investigated in order to elucidate the enzyme-induced distortion of the substrate at the +1 and -1 positions. TseI is a thermophilic type II restriction enzyme which has ideal activity at an elevated temperature. It is able to recognise and cut the 5 bp palindromic sequence of 5’-GCWGC-3’ (W=A or T). A range of biophysical methods have been applied to investigate this enzyme, including size-exclusion chromatography; fluorescence anisotropy (Kd value determination); denaturing HPLC for DNA cleavage analysis on matched and mismatched substrates; fluorescence-based activity assay (KM, Vmax, kcat, specificity constant values determination); steady-state fluorescence measurements (DNA-enzyme interaction study). The DNA cleavage characteristics of TseI were fully studied and it was found that it cuts A:A and T:T mismatches in CAG and CTG repeats. This potentially makes it a useful tool for exploring unusual DNA structures containing super-long CAG and CTG repeats which are involved in the aetiology of some neurodegenerative diseases, such as Huntington’s disease (HD). EcoP15I is a type III restriction-modification enzyme whose recognition sequence is 5-CAGCAG-3’. Methyltransferase EcoP15I (M.EcoP15I) adds a methyl group to the second adenine, in the presence of cofactor S-adenosyl methionine (SAM). SDS-PAGE, densitometry and size-exclusion HPLC were applied to confirm that EcoP15I adopts a Res1Mod2 stoichiometry in solution. The large structural distortion of its substrate (base flipping) by M.EcoP15I was investigated by both steady-state and time-resolved fluorescence. Also, nine 120 mer DNA duplexes, each containing two reversely oriented recognition sites were used to study matched and mismatched sequence cleavage by R.EcoP15 and a cleavage pattern was revealed.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental pollutants which are of concern due to their potential human toxicity. They are formed during numerous combustion processes, including biomass burning and diesel vehicular emissions, which are of relevance in developing countries. A novel analytical screening method for atmospheric polycyclic aromatic hydrocarbons (PAHs) was developed in this study based on laser induced fluorescence (LIF) of samples on quartz multi-channel polydimethylsiloxane (PDMS) traps. A tunable dye laser with a frequency doubling crystal provided the excitation radiation, and a double monochromator with a photomultiplier tube detected emitted fluorescence. The method allowed for the rapid (<5 min), cost effective analysis of samples. Those yielding interesting results could be further analysed by direct thermal desorption-gas chromatography-mass spectrometry (TD-GC-MS, with limits of detection of ~0.3 ng.m-3), as photodegradation was minimal (<10% over 5 min irradiation). Without any signal optimization, a LIF detection limit of ~1 ìg.m-3 was established for naphthalene using a diffusion tube (diffusion rate of 2 ng.s-1) and 292 nm excitation. Gas standards which facilitated the uniform distribution of analyte across each of the 22 PDMS tubes were provided by easily constructed diffusion tubes for naphthalene and by a gas chromatographic fraction collection method for the less volatile target PAHs. The methods developed were successfully tested in a number of applications which are of relevance to southern Africa, as emissions from sugar cane burning, household fires, diesel vehicles and industries were monitored. The LIF method allowed for the differentiation between impacted and non-impacted industrial sites, and the importance of naphthalene as an indicator for atmospheric PAHs was verified in that this PAH was the most abundant in the various applications which were investigated. The multi-channel silicone rubber traps were also evaluated theoretically and practically in the denuder configuration, in order to monitor PAHs in both the gas and particle phases, which is important in terms of human health effects. The novel LIF method developed in this study has the potential to serve as a screening tool to avoid the comprehensive and costly analysis of samples which do not contain appreciable levels of PAHs. The experimental procedure is simple and rapid, with acceptably low limits of detection, even with the initial, unoptimized optical arrangement and without extensive time-averaging. LIF also provides selectivity without the need for sample clean-up and separation processes. The LIF method could be further optimized by improving the laser energy stability, as well as by the investigation of possible time resolution techniques. As equipment cost considerations were important, it is possible that the LIF screening method could find application in a centralized environmental laboratory for the southern African region. This would facilitate the widespread monitoring of atmospheric PAHs in a cost effective manner. / Thesis (PhD)--University of Pretoria, 2010. / Chemistry / unrestricted