The rescue of blocked replication forks is vital to the faithful duplication of a cell's entire genome once per cell cycle. Eukaryotic cells are equipped with the intra-S phase checkpoint that co-ordinates the multitude of cellular responses to replicative stress, and evidence implicate the checkpoint in reflating fork restart by recombination pathways. In this study, I investigate the role of various Schizosaccharomyces pombe checkpoint gene products in the regulation of recombination at forks arrested by a programmed protein barrier, Replication Termination Sequence 1 (RTS1).

This work describes the interactions of small molecules with nucleic acid structures. Chapter 1 is concerned with the structure of DNA and goes on to explain the , structure of the DNA Holliday junction, an Intermediate in homologous recombination. Chapter 2 explains why the DNA Holliday junction is a potential therapeutic target for small molecules in the disruption of certain pathways utilizing homologous recombination. Chapter 3 goes on to describe the process of ligand design with the development of the bls(9-amlnoacrldlne-4carboxamides) as potential junction binders. Chapter 4 is concerned with DNA crystallography as a method to exami small molecule binding to nucleic acid structures.

In this thesis a literature review relating to the study of ligand-DNA interactions and he theory, principles and application of capillary electrophoresis (CE) for the study of such interactions is presented. A capillary polymer electrophoresis (CPE) method using, a hydroxypropyl cellulose (HPC) solution as sieving matrix, was developed to separate and measure ligand-DNA interactions.

This thesis is concerned with the rational design of a group of DNA higher order architectures known as quadruplex DNA. Quadruplex DNA is comprised of a stem of stacking guanine tetrads linked by loops comprised of single stranded DNA. Due to the different combinations of loop types possible, it has great structural diversity and has potential nanotechnological and biological applications. Currently, only a few loop combinations, or topologies are known. Those that have been determined experimentally were not explicitly designed. Using a geometric formalism, the sum of currently available knowledge on quadruplex folding was used to create a set of parameters for the design of novel quadruplex architectures. We present the proof of principle for rational design of said structures, through making the novel topology (-pd+l) and the (G:C:G:C) tetrad- containing topology -(Ppp). We apply this principle to interpret thermal difference spectra signatures of a number of designed quadruplexes. Finally, we show that such novel quadruplexes can be used as building blocks for nanowires. This thesis therefore describes the proof of principle of design, means to expediate design, and an example application of design, of quadruplex nucleic acids. Through achieving these three aims, we present the feasibility of designing novel quadruplexes to be used as nanotechnological or medical devices.

Gene regulatory regions, such as promoters or introns in the human genome, are rich in putative quadruplex forming sequences. G-quadruplex formation in these duplex regions requires the opening of duplex so that the single stranded region can fold into a G-quadruplex. The factors that affect the transition of duplex to G- quadruplex are currently not well understood. In this dissertation, we show that the availability of fragile sites, percentual GC content of the flanking sequence, loop composition, and loop-length, are all determinant factors for G-quadruplex formation within the native double helical segments. These factors are introduced and discussed in the first Chapter. In the following Chapters, 2 through 5, we investigate the propensity for formation of G-quadruplex topologies within the proximal promoter region of SRC, FGF2, and MYCN, as well as the first intron of IGF2. Except for MYCN we were able to establish formation of parallel quadruplexes from within the native double helical segments. We were not able to conclude on formation of a quadruplex in MYCN; even though there is evidence that a DNA higher order architecture forms. Significantly, the investigated guanine-rich segment of the proximal promoter of FGF2 gene shows propensity to fold into the parallel stranded quadruplex in the absence of crowding agents. In the final Chapter (6) these findings are discussed in the context of the factors that allow for the transition from closed to open double helix, and formation and stability of the parallel stranded quadruplexes. Finally, in this Chapter the biological significance of finding parallel stranded quadruplexes in this set of genes is discussed.

The last 15-20 years have seen an explosion in genome sequencing projects producing an unprecidented amount of data which supports the Identification and mapping of many genes. However, it does not describe their roles within the cell and functional genomics has attempted to discover uncover what these genes do with respect to their biochemical function, (sub)cellular localisation and processes m which they work. However, large proportions of genes within genomes still remain functionally uncharacterised, typically one third of a genome. Common homology-based approaches are arguably at their limit, and it is timely to consider the potential for alternative techniques to inform on gene function.

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G-quadruplexes (G4) are four-stranded DNA secondary structures, which are involved in a diverse range of biological processes. Although the anti-cancer potential of G4s in promoters of oncogenes has been thoroughly investigated, the functions of promoter G4s in non-cancer-related genes are not well understood. The aims of this thesis are (i) to investigate the biological importance of promoter G4s in cardiac function-related genes, and (ii) to explore the feasibility and potential of multitargeting strategy at the single gene level. These aims have been achieved by revealing the prevalence of G4s in TRRs (transcription regulatory regions) of cardiac function-related genes via bioinformatics approaches, and then investigating potential biological significance of G4s in promoters of human MEF2D and TnIc genes via a combination of biophysical, molecular, and cell biology approaches. By using bioinformatics approaches, TRRs of cardiac function-related genes were found to be enriched by potential G4-forming sequences. According to these results, G4s from the promoters of human MEF2D and TnIc were chosen and subjected to biophysical characterisations in solution. By using EMSA, DMS footprinting, CD and smFRET spectroscopy, the formation, thermodynamic stability, and unfolding kinetics of MEF2D and TnIc G4s were investigated. Briefly, in 100 mM K+, the MEF2DG4 can adopt a hybrid of very stable parallel/anti-parallel G4(s), while the TnIc MNSG4 and -80G4 are able to adopt anti-parallel and parallel structures, respectively, with comparable stability as compared to other oncogene G4s. Subsequently, cooperative regulatory roles of TnIc G4s as enhancers once stabilised by a G4-binding ligand (di-copper complex) were observed in vitro mainly by dual luciferase reporter assays in HEK293 cells. Three possible G4-mediated mechanisms have been proposed. This work provides the first indication about the biological significance of promoter G4s in cardiac function-related genes, and the feasibility to target multiple G4s at the single gene level.