This thesis covers different aspects of the evolution of the vertebrate beta globin gene family. A wealth of data on globins has been accumulated over decades of work in diverse areas, this information, together with the use of new methods, allowed a comprehensive analysis of beta globins. First, a review on the current knowledge of gene family evolution is made and the general objectives of the thesis are stated. This introductory chapter is followed by the careful analysis of the beta globin phylogeny comparing different reconstruction methods and discussing the differences between species and gene tree topologies. The molecular evolution of this gene family is investigated using codon models of sequence evolution. Particular emphasis is put on the role of gene conversion and positive selection acting at sites in the genes and along branches in the phylogeny. Also, several models of evolution by gene duplication are tested and results are analysed in the light of the different hypotheses on gene family evolution. The third chapter is devoted to the evolution of the globin protein structure from the analysis of sequence data. The ancestral state reconstruction of structurally relevant amino acids in different globins is conducted and the substitution pathway leading to the observed data is examined. The impact of amino acid changes in the hemoglobin protein is evaluated in terms of structural and functional constraints and the role of positive selection on the protein products of these genes is explored. Also, a possible case of coevolution between residues in the alpha and beta subunits of hemoglobin is proposed. Finally, using new and more sophisticated methods, I estimate dates for gene duplication and gene divergence events in the beta globin family. Two different methods of date estimation based on molecular data are compared and evolutionary rate variation in this gene family is tested.

p53 is the central protein in the DNA damage response and is part of a complex and extensive gene regulatory network. This network integrates a variety of stress signals to produce the up-regulation of active p53 and a range of effects including apoptosis, growth arrest and DNA damage repair. The p53 system has typically been studied qualitatively as a linear pathway, however this approach is insufficient to gain a full functional under standing of the dynamic nature of the network. In this work a better description of the DNA damage response will be constructed through the use of mathematical techniques. Ordinary differential equations models of the p53 network between DNA damage and p53 up-regulation are proposed, including a model that takes into account various localisation mechanisms. Parameter estimation is required to validate these models with biological data. A number of established techniques axe examined along with a novel method based on linear algebra, collocation and B-splines. To examine the network downstream of p53 and the global response to DNA damage, a "G" time profile (Gg(t)) quantifying the activity driving the formation of each gene is constructed. This is derived from a model of gene transcription, microarray data and mRNA degradation rates. The new parameter estimation technique developed works significantly better than the other techniques examined. Also, it was found that the mechanisms that control the location of p53 significantly contribute to the rapid DNA damage response. The G time profiles suggest that there are four principal transcription activities in the DNA damage response: p53, an early peaking response (possibly AP-1), stopping and restarting the cell cycle, and a double peaked response. The G time profile in combination with a training set of genes can be used to successfully find confirmed p53 targets.

It is hoped that the inhibition of the pathways that replicate and repair DNA will facilitate the development of new therapies in the fields of cancer treatment and antibiotics. For example, DNA ligases function in both DNA replication and repair pathways, sealing single-strand breaks in duplex DNA. The difference in cosubstrate specificity between the essential DNA ligases in bacteria and eukaryotes makes these enzymes potential drug targets for a novel class of antibiotics. Traditionally, DNA ligase activities have been measured using electrophoretic separation of DNA fragments to detect changes in the length of a reporter-labelled strand. However, this assay is slow and laborious with limited capacity. To allow screening of potential inhibitors and more extensive research in the field of DNA repair and replication, a new assay is required that has both higher-throughput and versatility in the experiments that can be performed. This thesis presents the development of such an assay using DNA hairpins labelled at one terminus by biotin and at the other by a fluorophore and immobilised in streptavidin coated microtiter plates through the biotin-streptavidin interaction. Loss or retention of fluorescence following denaturation of the hairpin reports on the integrity of the DNA backbone, which is altered by DNA ligase or nuclease activity. Using this immobilised DNA hairpin assay, the activities of the NAD+-dependent DNA ligases from Escherichia coli, Streptomyces coelicolor and Mycobacterium tuberculosis were studied. DNA ligase from bacteriophage T4 was also studied as a representative enzyme from the ATP-dependent class of DNA ligases. For the study of nuclease activity, restriction enzymes Alul, Ddel, KpnI, and Rsal were used, and the nicking enzyme N. BbvCI A. The activities of N. BbvCI A and DNA ligase from E. coli were combined to demonstrate sequential activity on a single sample of immobilised DNA hairpins. Such experiments provide a basis for reconstituting complete reaction pathways (such as DNA base excision repair) on single samples of immobilised substrate. The immobilised DNA hairpin assay was further employed tostudy the mixed activities of HeLa nuclear and whole cell extracts, and cell extracts from E. coli BL21 pLysS. Using the developed assay, the difference in activity between E. coli DNA ligase and the L15F variant is shown to be due to a much slower rate of enzyme adenylation in the latter. Finally, a theoretical framework to elucidate kinetic and mechanistic aspects of the immobilised DNA hairpin assay is presented. Thus, an assay is presented that can detect the activity of a wide range of DNA metabolizing enzymes in a format compatible with high throughput analyses. The assay is suitable for detecting activity both from purified enzymes and crude cell extracts. By exploiting the heterogeneous nature of the assay, novel experiments are possible that greatly facilitate analysis of DNA processing pathways when compared to the traditional methods.

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