A dissertation submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Master of Science. Johannesburg, 2013. / The forkhead box (FOX) proteins are a family of transcription factors that interact with DNA via a winged helix motif that forms part of the forkhead domain. The FOXP (FOXP1-4) subfamily is unique in the family in that the forkhead domains of these proteins exhibit domain swapping where structural elements are exchanged via extension of the hinge-loop region. The FOXP subfamily members have high sequence homology, yet wild-type FOXP3 is a stable domain-swapped dimer in solution whereas FOXP1 and FOXP2 exist in a monomer/domain-swapped dimer equilibrium. A single amino acid difference is observed in the hinge region of the FOXP subfamily. This corresponds to Tyr540 in FOXP2 and Phe373 in FOXP3. We propose that it is the phenylalanine residue in FOXP3 that shifts the equilibrium towards dimer. Here we use FOXP2 to investigate the effect of a mutation, Y540F, on the structure and dimerisation propensity of the FOXP subfamily. Crystals for the Y540F variant in the presence of DNA have been obtained to demonstrate conclusively that domain swapping occurs. Size-exclusion chromatography indicates that the wild type FOXP2 forkhead domain is almost entirely monomeric at concentrations less than 100 μM. The Y540F variant is shown to stabilise the dimer and the ratio between monomer and dimer is concentration-dependent. DNA binding assays suggest that the Y540F variant binds less favourably to the cognate binding sequence than does the WT FOXP2 forkhead domain. Taken together, these findings suggest that domain swapping may modulate DNA binding.

In eukaryotes, the genomic double-stranded DNA (dsDNA) coils around histones to form nucleosomes. Arrays of these nucleosomes bundle together to generate chromatin. Most DNA-related processes require interactions between chromatin-protected DNA and cellular machinery. Access of cell machinery to genomic DNA is partially regulated by the position and stability of nucleosomes, which may be influenced by changes in nucleosomal DNA. DNA is composed of adenine (A), guanine (G), cytosine (C), thymine (T) nucleotides and their derivatives. It has been shown that some C derivatives participate in directing multiple biological processes, and aberrant modification patterns are often linked to diseases. It has been proposed that T derivatives exhibit similar effects. This thesis focuses on elucidating the effect of naturally occurring DNA modifications on the properties of dsDNA and nucleosomes. dsDNA sequences systematically modified with various T derivatives were characterized using classical biophysical techniques to assess the effect of these DNA modifications. The results indicate that in the sequence context studied, 5-hydroxymethyluracil modifications destabilize dsDNA, while dense symmetrical 5-formyluracil (fU) modifications alter the dsDNA structure. These effects may provide clues to the differential protein recruitment observed in previous research. In vitro studies on nucleosome occupancy and stability revealed that 5-formylcytosine (fC) modifications have positive effects on nucleosome formation and stability compared to the unmodified counterpart by influencing the intrinsic biochemical and biophysical properties of the nucleosomes. These results provide casual links for the observation in vivo between fC and the increased nucleosome occupancy and positioning. In order to further understand the positional effect of fC on the nucleosomes, a method was developed for quick and reliable incorporation of C derivatives into dsDNA at desired positions. The positive effect of fC modifications on nucleosome occupancy and stability observed here has necessitated further studies to gain deeper insights into the biological functions of fC in the nucleosome context. Cryo-EM can be used to elucidate the structural foundation for the changes fC posts to nucleosome, and protein interacting assays will identify the cellular machineries specifically recruited/repulsed by fC-modified nucleosomes. The effect of DNA modifications elucidated by the above studies advances our understanding on the role that DNA modifications play in regulating cellular processes.

Graduation date: 1995

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Lai, Kin Fung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 92-97). / Abstracts in English and Chinese. / Title Page --- p.i / Thesis Committee --- p.ii / Abstract (In English) --- p.iv / Abstract (In Chinese) --- p.v / Acknowledgement --- p.vi / List of Figures --- p.xii / List of Tables --- p.xiv / List of Symbols and Abbreviations --- p.xvi / Chapter 1. --- Introduction --- p.1 / Chapter 1.1 --- Chemical Shift Predictions of Nucleic Acids --- p.1 / Chapter 1.1.1 --- Chemical Shift --- p.1 / Chapter 1.1.2 --- Chemical Shift Prediction of Double Helical DNA and RNA --- p.1 / Chapter 1.1.3 --- Chemical Shift Prediction of Random Coil DNA --- p.2 / Chapter 1.1.4 --- Applications of Nucleic Acid Chemical Shift Prediction --- p.4 / Chapter 1.2 --- General Review of DNA Structure --- p.4 / Chapter 1.2.1 --- Structure and Nomenclature of Nucleotide --- p.4 / Chapter 1.2.2 --- Structure of Polynucleotide --- p.5 / Chapter 1.2.3 --- Sugar Conformation in Nucleotide --- p.5 / Chapter 1.2.4 --- Double Helical DNA Conformation --- p.7 / Chapter 1.3 --- A.A Mismatches in DNA Duplexes --- p.8 / Chapter 1.3.1 --- Mismatches in DNA Duplexes --- p.8 / Chapter 1.3.2 --- Biological Significance of A． A Mismatches --- p.9 / Chapter 1.4 --- Purpose of the Work --- p.9 / Chapter 2 --- Materials and Method --- p.10 / Chapter 2.1 --- Overview of the Proposed Prediction Method --- p.10 / Chapter 2.1.1 --- Nearest Neighbor Model --- p.10 / Chapter 2.1.2 --- Base Pair Replacement Approach --- p.10 / Chapter 2.2 --- Sample Design --- p.11 / Chapter 2.2.1 --- Reference Sequences for Obtaining Triplet Values and Correction Factors --- p.11 / Chapter 2.2.2 --- Sequences for Verifying the Base Pair Replacement Approach --- p.12 / Chapter 2.2.3 --- Sequences for Testing Chemical Shift Prediction Accuracy --- p.12 / Chapter 2.3 --- Sample Preparation --- p.13 / Chapter 2.4 --- NMR Experiments --- p.14 / Chapter 2.4.1 --- Non-labile Proton Resonance Assignment --- p.14 / Chapter 2.4.2 --- Labile Proton Resonance Assignment --- p.16 / Chapter 2.5 --- Validating the Assumption in Reference Hairpin Model Samples --- p.17 / Chapter 3 --- Establishment of Proton Chemical Shift Prediction method of A.A Mismatches in B-DNA Duplexes --- p.18 / Chapter 3.1 --- Resonance Assignment --- p.18 / Chapter 3.1.1 --- Non-labile Protons --- p.18 / Chapter 3.1.2 --- Labile Protons --- p.20 / Chapter 3.2 --- Validating the Assumption in Reference Hairpin Model Samples --- p.21 / Chapter 3.3 --- Extraction of A.A Mismatch Triplet Chemical Shift Values --- p.22 / Chapter 3.4 --- Calculation of the 5´ة- and 3´ة-Correction Factors --- p.24 / Chapter 3.5 --- Chemical Shift Prediction Using Triplet Values and Correction Factors Extracted from Top Strands of refA.A(XAY) and refA．T(XAY) --- p.27 / Chapter 3.6 --- Chemical Shift Prediction Using Triplet Values and Correction Factors Extracted from Bottom Strands of refA.A(XAY) and refA．T(XAY) --- p.28 / Chapter 4 --- Testing of Proton Chemical Shift Prediction of A.A Mismatches in B- DNA --- p.29 / Chapter 4.1 --- Prediction Result Using Triplet Values and Correction Factors Extracted from the Top Strands of refA.A(XAY) and refA.T(XAY) --- p.29 / Chapter 4.2 --- Prediction Result Using Triplet Values and Correction Factors Extracted from Bottom Strands of refA.A(XAY) and refAT(XAY) --- p.30 / Chapter 4.3 --- Applicability of the Base Pair Replacement Approach --- p.31 / Chapter 4.3.1 --- Chemical Shifts and 3JH1´ةH2´ة of refT.A(XTY) Sequences --- p.31 / Chapter 4.3.2 --- Correction factors Extracted from the Top Strands of refA.A(XAY) and refT.A(XTY) --- p.31 / Chapter 4.3.3 --- Prediction Result Using Correction Factors Extracted from the Top Strands of refA.A(XAY) and refT.A(XTY) --- p.33 / Chapter 5 --- Conclusion --- p.35 / Appendix I NOE Sequential Assignment of refA.T(XAY) - (A) Aromatic Protons at 25 °C; (B) Labile Protons at 25 °C --- p.36 / Appendix II NOE Sequential Assignment of refA.A(XAY) - (A) Aromatic Protons at 25 °C; (B) Labile Protons at 5 °C --- p.40 / Appendix III H1'-H2'/H2´ح region of DQF-COSY Spectra of refA.T(XAY) at 25 °C --- p.44 / Appendix IV H1'- H2'/H2´ح region of DQF-COSY Spectra of refA.A(XAY) at 25 °C --- p.46 / Appendix V H3' region of HSQC Spectra of refA T(XAY) at 25 °C --- p.48 / Appendix VI H3' region of 1H-31̐ư HSQC Spectra of refA.A(XAY) at 25 °C --- p.50 / Appendix VII 3JH1'h2'1H and 31P Chemical Shifts of refA T(XAY) --- p.52 / Appendix VIII 3JH1'H2'and 31P Chemical Shifts of refA.A(XTY) --- p.60 / Appendix IX NOE Sequential Assignment of refT .A(XTY) - (A) Aromatic Protons at 25 °C; (B) Labile Protons at 25 °C --- p.68 / Appendix X H1'-H2'/H2''region of DQF-COSY Spectra of refT.A(XTY) --- p.72 / Appendix XI H3'region of H-31P HSQC Spectra of refT.A(XTY) --- p.74 / Appendix XII 3JH1'H2'1H and 31P Chemical Shifts of refT.A(XTY) --- p.76 / Appendix XIII Chemical Shifts of Testing Sequences --- p.84 / Reference --- p.92

In this dissertation, the formation of unusual G-quadruplexes in the critical regions of the c-myb and hTERT promoters for control of promoter activity was investigated.The c-myb promoter contains three copies of an almost perfect (GGA)4 sequence. We demonstrate that the each (GGA)4 repeat forms a tetrad:heptad G-quadruplex and any two of the three can intramolecularly dimerize to form T:H:H:T G-quadruplexes. The three T:H:H:T G-quadruplex combinations are of differing degrees of stability and can be further stabilized by G-quadruplex interactive compounds. We also demonstrate that the c-myb G-quadruplex forming region is a critical transcriptional regulatory element and interacts with various nuclear proteins including MAZ (Myc Associated Zinc finger protein). The data from luciferase reporter assay show that the c-myb GGA repeat region plays dual roles as a transcriptional activator and an inhibitor by serving as binding sites for the activators and by forming G-quadruplex structures in the region, respectively. Furthermore, we show that MAZ is a transcriptional repressor of the c-myb promoter and binds to both the double-stranded and T:H:H:T G-quadruplex-folded conformations of the GGA repeat region of the c-myb promoter.The hTERT core promoter contains a G-rich region of 12 consecutive G-tracts, which includes three critical Sp1 binding sites. Although this G-rich region has the potential to form multiple G-quadruplexes, our investigation on the full-length G-rich sequence demonstrate that the G-rich region forms a unique G-quadruplex structure in which two tandem intramolecular G-quadruplex structures are present, consisted of one G-quadruplex formed by the G-tracts 1-4 and the other formed by the G-tracts 5, 6, 11, and 12. We also demonstrate that the latter unusual structure contains a 26-base middle loop that likely forms a hairpin structure and is more stable than the other conventional G-quadruplex. Significantly, the formation of this unusual tandem G-quadruplex structure in the full-length will disable all three critical Sp1 binding sites, which will dramatically downregulate hTERT expression. G-quadruplex formation in the hTERT promoter suggests that the effect of G-quadruplex interactive ligands on telomerase inhibition and telomere shortening may be exerted by the direct interaction between the hTERT G-quadruplex structure and the ligands.

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Previous evidence has shown that the simple sequences microsatellites and poly-purine/poly-pyrimidine tracts (PPTs) could be both a cause, and an effect, of meiotic recombination. The causal link between simple sequences and recombination has not been much explored, however, probably because other evidence has cast doubt on its generality, though this evidence has never been conclusive. Several questions have remained unanswered in the literature, and I have addressed aspects of three of them in my thesis. First, what is the scale and magnitude of the association between simple sequences and recombination? I found that microsatellites and PPTs are strongly associated with meiotic double-strand break (DSB) hotspots in yeast, and that PPTs are generally more common in human recombination hotspots, particularly in close proximity to hotspot central regions, in which recombination events are markedly more frequent. I also showed that these associations can't be explained by coincidental mutual associations between simple sequences, recombination and other factors previously shown to correlate with both. A second question not conclusively answered in the literature is whether simple sequences, or their high levels of polymorphism, are an effect of recombination. I used three methods to address this question. Firstly, I investigated the distributions of two-copy tandem repeats and short PPTs in relation to yeast DSB hotspots in order to look for evidence of an involvement of recombination in simple sequence formation. I found no significant associations. Secondly, I compared the fraction of simple sequences containing polymorphic sites between human recombination hotspots and coldspots. The third method I used was generalized linear model analysis, with which I investigated the correlation between simple sequence variation and recombination rate, and the influence on the correlation of additional factors with potential relevance including GC-content and gene density. Both the direct comparison and correlation methods showed a very weak and inconsistent effect of recombination on simple sequence polymorphism in the human genome.Whether simple sequences are an important cause of recombination events is a third question that has received relatively little previous attention, and I have explored one aspect of it. Simple sequences of the types I studied have previously been shown to form non-B-DNA structures, which can be recombinagenic in model systems. Using a previously described sodium bisulphite modification assay, I tested for the presence of these structures in sequences amplified from the central regions of hotspots and cloned into supercoiled plasmids. I found significantly higher sensitivity to sodium bisulphite in humans in than in chimpanzees in three out of six genomic regions in which there is a hotspot in humans but none in chimpanzees. In the DNA2 hotspot, this correlated with a clear difference in numbers of molecules showing long contiguous strings of converted cytosines, which are present in previously described intramolecular quadruplex and triplex structures. Two out of the five other hotspots tested show evidence for secondary structure comparable to a known intramolecular triplex, though with similar patterns in humans and chimpanzees. In conclusion, my results clearly motivate further investigation of a functional link between simple sequences and meiotic recombination, including the putative role of non-B-DNA structures.