Our ever increasing knowledge of genetics is radically changing disciplines in science and medicine. Significantly, the study of gene expression and protein synthesis within both healthy and abnormal cells has advanced understanding of the mechanism of disease at the molecular level. The future treatment of certain diseases may benefit from new classes of nucleic acid based drugs which are currently undergoing development and trialling. Concurrently, assays are being formulated to predict, diagnose and monitor medical conditions. This more detailed patient analysis brings the option of moving away from traditional, textbook treatments and tailoring therapies to the individual, the field of personalised medicine. Current polynucleotide analysis platforms allow testing for genomic mutations and quantification of gene expression on a massively multiplexed scale with some arrays able to identify more than a million unique target sequences in a single assay. However much development is required to take this analysis technology from laboratory based applications to the bedside. Reductions in assay costs and analysis time are particular concerns. The 4G research group, based at the University of Southampton has developed novel encoded microparticle technology, allowing individual particles to be identified in a mixture. The work herein documents the adaption of this technology for the multiplexed analysis of DNA samples in the form of a suspension/hybridisation assay, a design which may offer advantages over current analysis technologies including reduced assay time and increased array flexibility.

Porphyrin moieties were rigidly attached to DNA to generate an accurate molecular ruler. Molecular ruler analysis was conducted using steady-state fluorescence, circular dichroism and small angle X-ray scattering spectroscopic techniques, in an attempt to analyse the FRET, exciton coupling and scattering intensity between different porphyrin-porphyrin labelled DNA combinations. A 21-mer test sequence was labelled with a porphyrin in one position on one strand, and seven different positions on seven complementary strands, to overall give seven porphyrin-porphyrin inter-strand combinations. Steady-state fluorescence and circular dichroism spectroscopic analysis of the Soret band revealed individual Watson-Crick bases pair molecular ruler sensitivity. Small angle X-ray scattering attempts between metallated-porphyrin entities did not reveal sufficient scattering at low concentrations, in contrast, an iodinated analogue of the porphyrin system did displayed scattering correlating to different iodine iodine distances. After calibration of the porphyrin system, the moieties were applied to study protein-DNA interactions between Tus, a 36 KDa DNA binding protein, and Ter, a specific 21-mer DNA sequence. Molecular ruler nalysis of the complex required an extended version of the Ter DNA sequence to which modifications were attached. Established FRET pairs FAM and TAMRA were applied to investigate protein-DNA complexation. Native PAGE analysis revealed Tus binds to the extended DNA via a sliding mechanism. Fluorescence analysis of the established FRET pairs identified changes in fluorescence not correlating to changes in FRET, and instead was attributed to emission quenching upon protein binding. Applying the zinc and free base porphyrin version displayed subtle changes in the Soret band circular dichrosim upon complexation, indicating small DNA helical change upon complexation. A 45-mer DNA sequence was designed to form multiple hairpin-duplex conformations with the addition of an appropriate complementary strand. Attaching FRET pairs to the extremes of the DNA sequence enabled multiple DNA conformations, and hence FRET distances to be obtained from one doubly modified DNA sequence. The combinations were characterised by UV-Vis, fluorescence and circular dichroism spectroscopy. Finally, terpyridine labelled DNA sequences selectively formed DNA nanotubes through orthogonal hydrogen bonding and metal complexation interactions. Short DNA strands were designed to self-assemble into long duplexes through a sticky-end approach. Addition of weakly binding metals such as zinc induced the formation of tubular arrays consisting of DNA bundles 50-200 nm wide and 2-50 nm high. TEM displayed additional long distance ordering of the terpyridine-DNA complexes into fibers.

The mitotic cell cycle underlies propagation of eukaryotic cells, continually duplicating and dividing. The past few years have seen major advances in understanding of the regulatory mechanisms that impose on the cell cycle to tightly co-ordinate progression through its individual phases, safeguarding the timing and integrity of its hallmark events, DNA synthesis and mitosis. Transcription is prominent among these processes, manifesting its importance for cell cycle controls by the large number of eukaryotic genes that are expressed at specific cell cycle times. Certain genes are cell cycle regulated in a number of organisms, suggesting that their phase-specific transcription is important for all eukaryotic cells. The budding and fission yeasts, Saccharomyces cerevisiae and Schizosaccharomyces pombe, have been used extensively as model organisms for the study of the eukaryotic cell cycle and cell cycle-regulated transcription, because the cell cycle machinery is conserved among eukaryotes and they are experimentally tractable. Recent microarray analyses have shown that cell cycle-specific expression is a frequent theme in the two yeasts, identifying consecutive, inter-dependent, waves of transcriptional activity, coinciding with the four main cell cycle transitions; G1-S, S, G2-M and M-G1 phases. Each phase-specific transcriptional wave corresponds to at least one group of co-regulated genes, sharing common cis- and trans- acting elements. The work presented in this thesis delves into the regulatory network that drives phase-specific gene expression during late mitosis-early G1 phase in fission yeast. During this late cell cycle stage, fission yeast and, indeed, every eukaryotic cell, undergo major changes; each completes mitosis and cytokinesis, partitioning its duplicated genetic and cytoplasmic material into two progeny cells, which then themselves prepare for a new round of mitotic cell division. Consistent with their periodic pattern of expression, most of the genes transcribed during the M-G1 interval in S. pombe encode proteins that execute important functions during late mitosis and cytokinesis. A DNA sequence promoter motif, the PCB (Pombe cell cycle box), has been identified in fission yeast that confers M-G1 specific transcription, and is bound by the PBF (PCB binding factor) transcription factor complex. PCB promoter motifs are present in several M-G1 transcribed genes, including cdc15+, spo12+, sid2+, fin1+, slp1+, ace2+, mid1+/dmf1+ and plo1+, the latter encoding a Polo-like kinase that also regulates M-G1 gene expression and influences the PCB-dependent binding properties of PBF. Three transcription factors, Sep1p and Fkh2p, both forkhead-like transcription factors, and Mbx1p, a MADS-box protein, have been implicated in M-G1 specific gene expression and are thought to be components of PBF. Consistent with Fkh2p and Sep1p regulating M-G1 specific transcription, forkhead-related sequences are present in the genes’ promoters. Notably, fkh2+ contains both PCB and forkhead promoter sequences and is transcribed during the M-G1 interval, implying that Fkh2p and Plo1p regulate gene transcription during late mitosis and ensuing passage through cytokinesis via feedback loops. This study provides further evidence about transcriptional regulation late in the fission yeast cell cycle, revealing that the PCB sequence is crucial for M-G1 specific transcription, with forkhead-associated DNA motifs playing a parallel but smaller regulatory role. Consistent with this hypothesis, work here and elsewhere shows that both Fkh2p and Sep1p control phase-specific expression of their co-regulated genes through the PCB and forkhead sequences. Notably, data in this thesis reveal that these two forkhead transcription factors associate with each other in vitro and in vivo and bind in vivo to the PCB promoter regions of M-G1 transcribed genes, including cdc15+ and plo1+, in a cell cycle specific manner, consistent with Fkh2p repressing and Sep1p activating transcription. Furthermore, Fkh2p contacts its own promoter, suggesting that it regulates its own expression via a negative feedback mechanism. The Plo1p kinase is shown here to bind in vivo to Mbx1p throughout the cell cycle and in a manner that requires both its kinase and polo-box domains. In agreement with this observation, Plo1p can phosphorylate in vitro Mbx1p, itself known to become phosphorylated during late mitosis. This is the first time that a Polo-like kinase has been shown to bind and phosphorylate a MADS-box protein in any organism. Moreover, in concert with Plo1p binding and phosphorylating Mbx1p, ChIP assays here reveal that this kinase interacts in vivo with the PCB promoter DNA of M-G1 expressed genes, including cdc15+ and fkh2+, in a cell cycle-dependent manner with a timing that coincides with low levels of expression, but follows promoter binding by Fkh2p. Given that Plo1p has previously been shown to positively influence M-G1 dependent transcription, its cell cycle pattern of promoter contact suggests that this Polo-like kinase functions at the genes’ promoters, most-likely via binding and phosphorylation of Mbx1p, to re-stimulate transcription, following repression by Fkh2p. In parallel, these findings suggest that Plo1p regulates its own expression via a positive feedback loop. Overall, the work presented in this thesis unravels crucial regulatory aspects of the transcriptional network that drives M-G1 specific transcription in S. pombe: it suggests an important role for the PCB promoter motif in transcriptional regulation; it proposes that Fkh2p acts as a repressor while Sep1p as an activator of late mitotic transcription; it reveals and proposes novel functions for Plo1p, a conserved Polo-like kinase family member, involving its association with Mbx1p, a MADS box protein, and its cell cycle specific recruitment to PCB promoters of M-G1 transcribed genes. As transcriptional systems, encompassing homologues of most of the components of this S. pombe M-G1 specific transcriptional network operate both in S. cerevisiae and humans, this demonstrates their importance for mitotic cell cycle progression. Thus this work potentially offers new insights into M-G1 specific gene expression in all eukaryotes.

The re-emergence of plague as a world-wide health concern and the potential risk posed by bioterrorism has led to an increased interest in available treatments for the disease. The bacterial DNA adenine-N6 methyltransferase, Dam, is involved in the regulation of a range of pathogenic bacteria and has been validated as a target for the development of antimicrobial agents with activity against Yersinia pestis, the causative agent of plague. The lack of a functionally similar enzyme in mammals suggests that highly selective Dam inhibitors could be developed. A coupled, real-time break light Dam activity assay has been optimised for HTS, and assays for the validation and characterisation of screening hits have also been developed. Screening of random and in silico enriched compound libraries, and the subsequent application of counter-screening and hit confirmation assays, resulted in the identification of a single viable lead, (4-(N-(2-hydroxyethyl)sulfamoyl)phenyl) stibonic acid (13776). Screening of compounds analogous to 13776 identified a series of arylstibonic acids with activity against Dam. Kinetic characterisation of the most potent arylstibonic acid, 4-stibonobenzenesulfonic acid (13746), revealed a DNA-competitive mode of action, and a Ki of 6.46 ± 0.07 nM. However, selectivity assays have revealed a potentially non-specific mode of action for the stibonic acids, which have shown activity against a range of DNA and protein binding enzymes. Yersinia cell culture experiments have shown a single compound, (3-((2-hydroxyethyl)carbamoyl)phenyl)stibonic acid (13782), to be capable of penetrating Yersinia cells and partially inhibiting methylation, and mRNA profiling experiments have shown 13782 to induce a statistically significant change in several genes involved in the pathogenicity of Y. pestis. Attempts at resynthesising 13782 have proved challenging, with only a fraction of the activity of the original sample reproduced. HPLC analysis of the original and resynthesised samples has shown the former to comprise two components, with only one present in both samples. The in vitro evaluation of a series of bisubstrate analogues designed to mimic both the methyl donor S-adenosylmethionine (AdoMet), and the methylation target (adenine) has shown that substitution of the AdoMet sulfur for nitrogen results in a significant but not total loss of activity. Furthermore, the addition of a bicyclic heteroaromatic adenine analogue mimic to this scaffold led to an increase in potency and selectivity for Dam over the human cytosine methyltransferase DNMT1 but a reduction in selectivity for Dam over the restriction enzyme DpnI. These results suggest that a selective and potent Dam inhibitor can be obtained by carefully modifying both components of the bisubstrate analogue inhibitor.

Chapter 1 reviews DNA structure and DNA molecular recognition by synthetic agents, including supramolecular helicates and cisplatin. An overview of various types of helicates and cisplatin anticancer drugs and their mechanisms of action is discussed. Examples of non-platinum anticancer drugs are also presented. In Chapter 2, the synthesis of novel metal-based supramolecular helicates and attempts to combine them with the cisplatin anticancer agent are described. In some cases X-ray crystallography data are presented and discussed in detail. In addition, the synthesis of the fluorescent europium helicate is described. In Chapter 3, stability and DNA binding properties of the synthesised metallo-helicates are investigated using UV/Vis spectroscopy, CD and LD techniques. The ability of the complexes to unwind plasmid DNA and stabilise DNA three-way junction formation is explored by gel electrophoresis experiments. The results demonstrate that the geometry and size of the helicates are crucial for DNA three-way junction recognition. In Chapter 4, biological evaluation of antineoplastic activity of the metallo-helicates synthesised in this work, as well as of the compounds supplied by other members of the group, is studied using an MTT colorimetric assay. The results of the study reveal that some of the complexes exhibit a potent cytotoxic activity.

In order to study computationally increasingly complex systems using theoretical methods model Hamiltonians are required to accurately describe the potential energy surface they represent. Also ab-initio methods improve the calculation of the excited states of these complex systems becomes increasingly feasible. One such model Hamiltonian described herein, the Vibronic Coupling Hamiltonian, has previously shown its versitility and ability to describe a variety of non-adiabatic problems. This thesis describes a new method, a genetic algorithm, for the parameterisation of the Vibronic Coupling Hamiltonian to describe both previously calculated potential energy surfaces (allene and pentatetraene) and newly calculated (cyclo-butadiene and toluene) potential energy surfaces. In order to test this genetic algorithm quantum nuclear dynamics calculations were performed using the multi-configurational time dependent hartree method and the results compared to experiment.