Methods to Probe the Function of Modified Bases in DNA

This thesis is focused on the development and utilisation of chemical and biological tools to probe the function of modified bases in DNA with specific exploration of the less well-studied T-modifications: 5-hmU, 5-fU and Base J. LCMS/MS techniques are first utilised to enable the accurate global quantification of T-modifications (5-hmU, 5-fU and Base J) in both trypanosomatids and mammalian DNA. A chemical affinity-enrichment sequencing method for the T-modifications is next described, which allows their chemoselective tagging over their C-modification counterparts. DNA fragments containing 5-fU are selectively tagged and enriched via oxime, hydrazine or benzimidazole formation using a biotinylated probe, and DNA fragments containing 5-hmU can be first chemically oxidised to 5-fU using KRuO4. .Proof-of-principle T-modification enrichment is demonstrated by DNA sequencing. In the following chapter, sequencing methods are employed to investigate the role of T-modifications in both trypanosomatids and mammalian samples. In T.Brucei, Base J formation is probed by artificial incorporation of 5-hmU and subsequent Base J chemical sequencing. Base J is preferentially formed or depleted at certain genomic loci; suggesting that Base J formation is sequence-specific. This may imply a distinct role for the 5-hmU sites which are not further glucosylated. Next, 5-hmU enrichment sequencing is performed in SMUG1 knockdown HEK293T cells to determine the genomic location of 5-hmU in mammals. An increase in 5-hmU loci is observed upon SMUG1 knockdown. 5-hmU enriched regions are found to be T-rich and depleted in exons and promoters. Furthermore, 5-hmU sites show poor overlap with known TET-enzyme binding sites, indicating that 5-hmU is formed via a TET-independent mechanism in HEK293T cells. Next, mass spectrometry-based proteomics studies are utilised to determine 5-fU protein-binders in mammals. Pulldown of proteins using biotinylated baits enables the identification of proteins which are enriched or suppressed in the presence of the 5-fU modification compared to a non-modified control. Enriched proteins include those associated with DNA-damage, consistent with the current understanding that 5-fU is a product of oxidative damage in mammalian DNA. Finally, a mechanistic insight into the effect of formylated bases on nucleosomal structure is described. Schiff base formation between formylated nucleobases and histone protein lysine side-chains is demonstrated. This provides a molecular mechanism for the association of 5-fC with increased nucleosomal occupancy in vivo.CHESS scholarshi

Lifeways at the Mesolithic-Neolithic Transition: Integrating New Biomolecular Approaches to Skeletal Material in Britain

The Mesolithic-Neolithic transition is a period which has long held fascination for archaeologists, and yet the lifeways of individuals at this time are still not fully understood – in part due to the lack of human remains in Britain from the period. This thesis therefore aimed to adopt a combined biomolecular approach to determine more information about the lifeways of both the Mesolithic and Neolithic of Britain, and of the transition between them, but utilising archaeological material not traditionally included within these debates – notably unidentifiable bone fragments, disarticulated skeletal remains, and dental calculus. Through analysis of these materials, the thesis focuses on five main areas of interest: identification, diet, mobility, chronology, and health/disease; utilising six different techniques: ZooMS, δ13C and δ15N stable isotope analysis, 86Sr/87Sr isotopic analysis, AMS dating, and metagenomic and metaproteomic analysis of dental calculus. As such, this marks the largest combined application of biomolecular techniques to British Mesolithic and Neolithic material to date. The results of this study highlight the value which may be held within previously overlooked early prehistoric archaeological materials, and the information which they may be able to contribute to existing discussions of Mesolithic and Neolithic lifeways. Overall, it can be seen that the main outcomes of this study are (i) that additional human remains may be present within early prehistoric ‘unidentifiable’ fragmented bone assemblages; (ii) dietary complexity in both the Mesolithic and Neolithic of Britain may be greater than previously thought; (iii) enhanced understanding of Neolithic mobility; (iv) a reconsideration of the approach to chronology at the Mesolithic-Neolithic transition; and (v) that dental calculus may provide a suitable and useful new medium via which to study prehistoric health and disease in future studies