Advances in molecular engineering have enabled the formation of increasingly sophisticated molecular systems. Use of DNA and other biomolecules has proven a particularly powerful tool for nanotechnology, with their unique chemistry allowing the synthesis of self-assembling nanoscale devices with complex structures and functionalities. The ability to integrate such constructs with solid state electronic devices would be of great value for the development of these technologies into practical devices. In this project, a method was developed allowing the specific targeted alignment and binding of single molecules to sites on nano-patterned metal electrodes, relying on the highly specific molecular recognition capabilities of DNA. The patterning method utilised self-assembled monolayers of I-mercapto11- undecanol as a molecular resist, which could be removed via reductive electrochemical desorption of the gold-thiol bond. This allowed the patterning of thiolated DNA probes on selected electrodes in an array. A DNA strand with sticky ends complementary to the surface probes can then specifically bind to the surface, bridging between sites where this enables the simultaneous hybridisation of both its single stranded regions. The surface binding and hybridisation of thiolated DNA oligonucleotides was tested using a colorimetric surface staining technique and the quality of monolayers was investigated using several methods. These trials informed the development of DNA-coated surfaces resistant to non-specific binding. The electrochemical desorption of SAMs was then investigated as a means for the high-resolution patterning of surfaces. Employing these techniques, the specific bridging of gold electrodes separated. by 70nm with 330 basepair DNA strands was demonstrated. Additionally, the selective thermal melt ing of different DNA probes and the ligation of surface-bound DNA constructs were examined as further methods of controlling the specificity of the assembly reaction.

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DNA double strand breaks arise endogenously in the cell as a result of routine activities such as metabolism and also as a result of exposure to exogenous agents such as ionising radiation. Repair of DNA DSBs is coordinated by two major repair pathways in mammalian cells; non-homologous end joining and homologous recombination. Nonhomologous end joining is the dominant repair pathway during G1 and G2 phase of the mammalian cell cycle. The core non-homologous end joining factors Ku, DNA-PKcs, XLF, DNA Ligase IV and XRCC4 are essential for efficient and accurate rejoining of double strand breaks. In 2004, Riballo et al. discovered that the ATM protein kinase and the Artemis endonuclease were important for repair of a fraction of DNA double strand breaks in G1 phase cells.

Previous workers have shown that high magnetic fields cause morphological changes in DNA at high concentration where it exists as the liquid crystal cholesteric phase. The aim of the present work was to identify corresponding changes in the dielectric properties of DNA in the frequency range 20 Hz to 300 kHz, at lower magnetic field intensity and lower concentrations. Investigations were carried out with DNA solution on interdigitated electrodes, where the magnetic field was applied perpendicular to the electric field. Evidence was found of reversible changes in the dielectric properties caused by magnetic fields lower than 0.7 Tesla at high concentrations (rv 170 mg/ml). Using planar electrodes, where the magnetic field was applied parallel to the electric field, reversible changes in dielectric properties due to magnetic fields of lower than 0.7 Tesla were found in DNA solutions at lower concentrations (25 - 100 mg/rnl). Replacement of the sodium in the DNA with other cations by dialysis produced similar results, though the frequencies of the responses also varied with the cations used. A method was developed using Matlab's Genetic Algorithm toolbox to analyse the difference between loss spectra caused by changes in one dispersion. The method was able to extract the parameters of the dispersion before and after application of the magnetic field. Changes in these parameters with concentration, temperature, additional sodium chloride, and applied a.c.voltage are reported. Changes with temperature indicate that an Arrhenius-type process is responsible for the dispersion. Evidence was presented that the dispersion found to be susceptible to magnetic field was caused by relaxation of the counter-ions in the condensed phase surrounding the DNA chain.

DNA polymerases play key roles in DNA synthesis by catalysing the polymerisation of deoxynucleotides opposite a parental (template) DNA strand to generate a new or repaired complementary daughter strand. Faithful replication by DNA polymerases is essential in maintaining genome integrity during cell division, DNA repair and DNA recombination. DNA polymerase beta (Pol β) plays a pivotal role in the base excision repair (BER) pathway by performing repair synthesis to fill single nucleotide gaps which arise during DNA repair. However, overexpression of Pol β is found in many human cancers and has been shown to promote a mutator phenotype. The aim of this study was therefore to investigate the effect of Pol β overexpression on BER. In vitro repair assays using whole cell extracts and DNA substrates containing site-specific BER lesions were conducted to compare two cell lines, one of which was derived from a cancer patient overexpressing Pol β. I found that overexpression of Pol β results in a 5 to 10-fold increased frequency of one nucleotide frameshift mutations and based on biochemical studies a mechanism is proposed to explain this phenomenon. I therefore conclude that an excess of Pol β can have potentially mutagenic consequences.

Nucleic acids are being targeted more commonly as pharmaceutical therapeutic agents, and consequently the ability to purify these biological macromolecules at large scale has taken on increased importance. Although precipitation of nucleic acids plays a key role in existing purification processes, the effects of different process variables are not well understood. In recovery processes, typically supercoiled plasmid DNA is the form of interest, however because other nucleic acid forms (chromosomal DNA, RNA, relaxed plasmid DNA) are very similar to plasmid DNA both structurally and chemically, separation of plasmid DNA from these forms represents a significant challenge. This project studied the precipitation behaviour of different nucleic acids forms in typical clarified lysate streams from E. coli fermentation. Solubilities of individual forms were studied, investigating effects of process variables such as salt type, salt concentration, organic solvents, and polymers. Key findings from single-component studies showed monovalent cations alone to be relatively ineffective agents for precipitation of any of the nucleic acid forms studied. Divalent cations alone were relatively ineffective for precipitation of double- stranded DNA, however they were effective agents for precipitation of RNA and single-stranded DNA, suggesting these salts may be effective for fractional precipitation of these forms. Inclusion of organic solvents or polymers decreased solubilities of all forms in the presence of either monovalent or divalent cations. Single-component findings were extended to investigation of multi-component clarified lysate process streams. To aid in this effort, an analytical anion- exchange HPLC method was developed to quantify different nucleic acid forms from multi-component streams. This analytical method was used to show divalent cations were effective for the fractional precipitation of the majority of single-stranded nucleic acid impurities in clarified lysate, leaving most of the plasmid DNA plus small quantities of chromosomal DNA and RNA impurities in solution. Single-component results were further extended through the development of a novel controlled thermal-denaturation step prior to divalent cation precipitation. This step converted double-stranded DNA forms, with the exception of supercoiled plasmid, to the single-stranded form. Performing thermal denaturation on clarified lysate streams prior to divalent cation precipitation resulted in improved separation of chromosomal and relaxed plasmid DNA forms, leaving highly-purified supercoiled DNA in solution following precipitation. A non-chromatographic purification process for recovery of supercoiled plasmid DNA is proposed based on these results. Findings were further characterised through kinetics of precipitation studies, investigation of precipitation mechanisms, comparison to solubility models, and investigation of plasmid stability.

Genomic DNA is organised in complex spatial arrangements, and a given stretch of DNA may encode more than one gene. In some cases one gene may be entirely contained within a region of the DNA already occupied by another larger gene. The presence of these nested genes, often situated in introns and in the opposite orientation, poses important implications with regards to gene expression, function and regulation. A consequence of the nested gene arrangement is convergent transcription, occurring when two promoters on opposite DNA strands are active. Elucidating the mechanics of multiple interacting proteins on single DNA templates requires single molecule methods such as atomic force microscopy (AFM). AFM can accurately determine the relative positions of enzymes, such as RNA polymerase (RNAP), on individual DNA templates. The central aim of this thesis is to use AFM to study the outcomes of convergent transcription, using linear DNA templates that function as models for nested genes. Fundamental aspects of imaging DNA on mica with AFM were investigated, with a view to optimising sample preparation. The main processes involved with preparing DNA samples, ready for scanning, were examined in turn. Effective binding was achieved by introducing divalent cations into a deposition buffer. Mica ion exchanged with Ni(II) usually gave rise to kinetically-trapped DNA molecules, however short linear fragments (< 800bp) were seen to deviate from the expected behaviour, indicating that ion-exchanged mica is heterogeneous, and contains patches or domains. These findings can be used to more readily control binding of DNA to substrates. The outcomes of varying the relative humidity while imaging biomolecular systems are largely unexplored to date. Various DNA samples were imaged in conditions of varying humidity. In particular when supercoiled plasmids are scanned at very high relative humidity (> 90% RH), localised DNA backbone motions or conformational changes were observed. Humidity controlled AFM will be a useful technique for probing DNA topology without some of the drawbacks of imaging under bulk solution. Initial studies of transcription utilised templates containing two promoter sites and E. Coli RNAP. Two promoter arrangements were studied: a convergent template containing the promoter sites on opposite strands directed towards each other, and a tandem template containing the promoters in the same direction, on the same stand. It was shown that collisions between RNAPs led to similar outcomes in both cases: RNAPs are unable to pass each other and remain stalled against each other. In the convergent case, it was observed that after collision one RNAP could cause another to backtrack along the template. By end-labelling double-stranded (ds) DNA templates with a single-stranded DNA loop, the polarity of the molecules can be established in the AFM. It allowed better discrimination between outcomes of collision events on single DNA molecules and importantly, it enabled a quantitative comparison of the relative frequencies of the outcomes. The most common outcome is a collision between an actively transcribing elongation complex (EC) and a “sitting duck” (SD), which is a stalled RNAP or open promoter complex (OPC). In collisions initiated from OPCs, the most likely outcome, a collision between an EC and an SD occurs ~74% of the time. This causes sizeable back-tracking of the inactive RNAP, on average 59 nm upstream of the promoter. A significant fraction of the collisions (~15%) are between actively transcribing RNAP while the remainder (~11%) are undetermined. End-labelling of dsDNA using nucleic acid structures did not interfere with AFM sample preparation and can be used as a generic approach to studying interactions of multiple proteins on DNA templates at the single molecule level.

An efficient DNA damage response is critical for maintaining the integrity of the mammalian genome, and ensuring the accurate transfer of genetic information between generations. Of particular biological relevance are DNA double strand breaks (DSB), which if repaired incorrectly may contribute to carcinogenesis. Review of contemporary literature has led to the identification of protein interactions and transcriptional events, tightly associated with the mammalian DSB response. Characteristics of selected events have been manipulated, with the notion of developing a reporter system that offers a sensitive and rapid method of detecting DSB in living mammalian cell models. Work presented here provides a quantitative evaluation of DSB generation in various mammalian cell lines, following chemical and irradiation treatment, and highlights the limitations of currently used markers. A series of recombinant proteins comprising peptide interacting domains, which exhibit altered spatio-temporal dynamics in relation with each other following DSB induction, are proposed as potential reporters of damage in mammalian cells. Novel gene constructs have been engineered that encode these peptide interacting domains, sandwiched between fluorescence-resonance-energy transfer (FRET) capable proteins. DSB specific events are predicted to induce peptide interactions that may be tracked in real time, by monitoring alterations in the fluorescent properties of such a recombinant protein. In an alternative approach, the transcriptional up-regulation of RAD52 mRNA following DSB induction was extended to whole cells. Optimisation of a fluorescent molecular beacon probe complementary to mammalian RAD52 mRNA is described, and data obtained in mammalian cells following DSB induction supports the notion that RAD52 is actively transcribed as part of the DSB response.