During this thesis a new infrastructure of methods to template functional materials at specific sites in a DNA architecture using molecular recognition of specific DNA duplexes has been developed. The project focused on the use of naturally occurring duplex DNA and the Tollens’ reaction as a mild source of metal amenable to reduction in the presence of aldehyde functional groups. The advantages of this choice are: 1) The use of naturally-occurring DNA increases modularity; 2) The Tollens’ reagent acts as a mild selective metallising reagent that enables the reduction of metal ions at specific sites along DNA. Molecules such as Pyrrole-Imidazole polyamides were prepared and were tested to determine whether selective metallisation of DNA nano-architectures can be achieved by confining the nucleation and growth of silver nanostructures to defined regions along a DNA duplex.

The B. subtilis protein DnaD is an essential component of the chromosome replication machinery and a global regulator of DNA architecture, as it exhibits a unique DNA remodeling activity that opens up supercoiled DNA by forming higher order nucleoprotein structures. There are no homologues of this protein in gram negative bacteria and details of its molecular structure are at present limited to the crystal structures of the N-terminal domain from B. subtilis and G. Kaustophilus, and also crystal structures of the C-terminal domain of two proteins with unknown function from S. mutans and E. faecalis (Structural Genomics projects pdb codes 2zc2 and 2i5u respectively). In this thesis, the determination of the NMR structure for the DNA binding C-terminal domain of the B. sllbtilis DnaD protein is reported. This domain is composed of five helices and an unstructured C-terminal tail, helix I-IV form a well packed hydrophobic core and helix V, which is more extensive than assumed from sequence alignments, extends away from this core fold of the protein. NMR DNA titrations, in vitro mutagenesis and in vivo complementation experiments show that a highly conserved YxxxIxxxW motif, the final helix and a portion of the mobile Cterminal region make up the DNA binding module of B. subtilis DnaD and are all essential for cell viability. Sequence alignments of DnaD alone fail to identify two key elements of this DNA binding module, the poorly conserved sequences of the final helix and the C-terminal mobile segment. Sensitive Hidden Markov Models based techniques, show that the two structural domains found in DnaD are present in B. subtilis DnaB, a primosomal protein that also participates in replication initiation. Furthermore, these two proteins share the only strong sequence motif, the highly conserved YxxxIxxxW sequence that contributes to DNA binding.

My research has focused on developing an understanding of processes operating in and around the Xp/Yp telomere, combined with detailed analysis of the unusual properties seen immediately adjacent to it. To this end, 7 kb of telomere adjacent-sequence was analysed, revealing a SNP density, pi, of 1.73 x 10-3 across the first 3 kb, 2-fold above average genome level. Furthermore, the sequence analysis revealed a 1.9 kb tandem duplication in 86% of chromosomes. This included the minisatellite DXYS14, and both copies were found to be hypervariable, with pedigree analysis revealing mutation rates of 0.94% and 0.62% for DXYS14a and DXYS14b respectively, with double mutations often occurring.;Haplotype analysis revealed strong SNP associations across the entire 7 kb region. High-resolution crossover analysis was carried out using allele-specific PCR methods to selectively amplify recombinant sperm molecules, which indicated that the Xp/Yp telomere-adjacent DNA is recombinationally inert, with a rate of 2.4 x 10-6 cM Mb-1 calculated from 1.250,000 amplifiable molecules.;It was thought secondary structure may be implicated in both the high SNP density and low recombination rate seen, so analysis of t-loops and nucleosome phasing was carried out at the Xp/Yp telomere. While the efficiency of t-loop visualisation prevented specific targeting of the Xp/Yp telomere, preliminary nucleosome data suggest that nucleosomes are present, but not phased in this region.;Finally, analysis was conducted on the presence of the CTAGGG variant repeat at the Xp/Yp telomere. This variant repeat is presented at ∼7% of Xp/Yp telomeres, and in CEPH families, two telomeres containing arrays of CTAGGG were shown to display a ∼46% germline mutation rate, higher than the 0.6% rate estimated in telomeres not harbouring this repeat. It is possible that secondary structure may be responsible for the instability associated with this repeat type.

How might the process of meiotic crossing-over affect the evolution of sequences and genome structure? Much attention has been focussed on the notion that crossing-over modulates the efficacy of selection. Here, we consider how good the evidence for this is. Correlations between recombination and protein rates of evolution, commonly interpreted in the above framework, might be misleading for failing to remove the effects of covariates or misinterpreted by disregarding direct effects of recombination, such as biased gene conversion. Similarly, higher diversity commonly seen in highly recombining domains may not necessarily imply a connection between recombination and diversity. It could, for instance, reflect a covariance with mutation rate variability owing to replication timing effects. This thesis examines not only these links between recombination and gene evolution but in addition asks other recombination-centred questions. Is gene order evolution in part driven by recombination predisposing certain sites to rearrangement? How can we account for the genomic location of recombination? Does, for example, germline expression, recently suggested to predict low recombination rates in mammals, predict recombination rates in flies? With the exception of the second chapter where we investigate the relationship between double strand break formation, recombination and sequence divergence in Saccharomyces cerevisiae, my work considers Drosophila, making use of the 12 genomes resource. While an effect of crossover on divergence owing to more efficient selection cannot be ruled out, we demonstrate that premeiotic double strand breaks also predict slow evolution. Late replication, we show, is associated with increases in both divergence and variation but this does not undermine the recombination-diversity correlation. While recombination is associated with increased rearrangement rates, we find no evidence that germline expressed genes avoid recombination.

The research presented within this thesis is concerned with the design and synthesis of redox-active derivatives for the use as labels for DNA probes to be used in a commercial DNA detection assay. Chapter 1: Introduces the area of electrochemical DNA sensing, the methods used and the transducer derivatives used. Also included within this chapter is the discussion on the use of ferrocene and its derivatives as redox-active transducer moieties and their use in a DNA sensing capacity. Chapter 2: Introduces the sponsoring company, Atlas genetics, and the DNA detection assay they are developing for use in a point of care device (POC). The chapter also details the design, synthesis and electrochemical analysis via differential pulse voltammetry of mono-ferrocenyl derivatives for the use as redox-active labels for DNA sensing. The chapter outlines the development of labels containing a variety of functionality as well as possessing a range of oxidation potentials from 95 to 610 mV (vs Ag/AgCl). Chapter 3: Introduces and details the sensitivity issues between mono-ferrocenyl and di-ferrocenyl labels and the effect this could have on the commercial DNA detection assay. Within this chapter there are the details of the design, synthesis and electrochemical analysis via differential pulse voltammetry of the di-ferrocenyl labels. The chapter shows the synthesis of di-ferrocenyl labels containing a wide range of functionality on both the ferrocene core as well as the linker unit, with the labels processing a range of oxidation potentials from 242 to 500 mV (vs. Ag/AgCl). Chapter 4: Discusses the use of the labels designed and synthesised in chapters 2 and 3 in the commercial DNA detection assay developed by Atlas genetics. The development of the labels used as DNA probes in the DNA detection assay and their ability to be used in multiplex DNA detection assays are also described. Demonstrated within this chapter is the use of the labels synthesised in this thesis that give both a duplex between two different probes and also the development of a triplex assay using three different labels to detect for two different target DNAs as well as provide an internal control. Chapter 5: Introduces the synthetic methods towards the synthesis of 2-oxazolines and discusses their use within ring-opening reactions. This chapter details the optimisation of the ring-opening reaction of phenyl-2-oxazoline with a range of carboxylic acid derivatives. The synthesis of a range of 2-oxazolines with various functionality aimed towards the analytical detection of carboxylic acids through the direct conjugation with 2-oxazolines. The ring-opening reaction was found to tolerate a wide range of carboxylic acid derivatives as well as a variety of functionality on the 2-oxazolines such as ferrocene-2-oxazolines and also 3,5 – bis(trifluoromethyl)phenyl-2-oxazoline. The use of ferrocene-2-oxazoline allowed for the electrochemical detection of carboxylic acids and the 3,5 – bis(trifluoromethyl)phenyl-2-oxazoline allows for thedetection of carboxylic acids through its ring-opened form via 19F NMR. These two functionalised 2-oxazolines were used to further analyse the reactions viability as a possible analytical tool for the detection of carboxylic acids by carring out the ring-opening reaction conditions for the detection of ibuprofen from an over the counter tablet.

Potentially carcinogenic DNA damage that may be derived from dietary sources can be quantified using a number of methods. Some of the best of these methods are based on the use of antibodies that have high affinity and specificity for modified DNA bases. However, given the sometimes limited availability of antibodies there is a requirement for novel reagents that mimic the properties of the best antibodies. From a chemical perspective antibodies are `over engineered' and synthetic affinity reagents would recreate the optimal properties of the binding site with respect to selectivity and affinity. Phage display was used to identify amino acid residues that contribute to recognition of a diet derived DNA adduct 06-carboxymethyl-2'-deoxyguanosine. However, this information was not sufficient to synthesise a novel peptide reagent directly and it was decided to develop an approach whereby a library of compounds was synthesised which includes the characteristics expected within the binding site of an antibody. This library was based around cholic acid, a readily available scaffold molecule with suitable functionality compatible with linking amino acids and a fluorescent tag. A synthetic procedure based on the formation of stable oxime derivatives of cholic acid was optimised and a small library of pyrene-tagged trisubstituted cholic acid derivatives was characterised. The library was examined for its ability to bind to affinity columns containing immobilised 06-carboxymethylguanosine and there was some evidence that indicated specific binding of a subset of library molecules

By comparison of developmental processes and features across animal taxa, one can reconstruct ancestral states and gain a deeper understanding of how different developmental modes and body plans evolved. ANTP-class homeobox genes are an ideal starting point to study the diversification of animals due to their essential functions in developmental processes, genomic organisation, confinement to animals, and highly conserved domains, which permit relatively robust orthologue classification between species.

RecQ helicases maintain genomic stability by their exceptional ability to resolve many replication or recombination intermediates in addition to normal B-form DNA duplexes. Microorganisms such as E.coli, Saccharomyces cerevisiae, and Schizosaccharomyces pombe express only a single RecQ enzyme per species, while higher eukaryotes need more than one RecQ helicase to function.

The family B DNA polymerase from the euryarchaeote, Pyrococcus furiosus (PFU-Pol), has been shown to recognize template strand uracil and stall DNA synthesis four bases ahead, thus preventing the permanent fixation of the mutation. This recognition is due to the presence of a special pocket in the N-terminal domain of the enzyme. This study confirmed the recognition of hypoxanthine, the deaminated product of adenine, by PFU-Pol. Primer extension assays demonstrated that template strand hypoxanthine leads to stalling of DNA synthesis, four bases demonstrated that template strand hypoxanthine leads to stalling of DNA synthesis, four bases in front of the base, exactly the same position as seen for uracil.

The DNA cleavage activity of a range of aryldiazonium compounds was investigated using defined sequence DNA, either in the form of synthetic oligonucleotides or as a fragment derived from pBR322 plasmid DNA. The cleavage assay involved high-esolution denaturing electrophoresis of the ³²P-5 - endlabeled DNA targets.