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Methods to Probe the Function of Modified Bases in DNA 10.17863/CAM.18804 Hardisty, Robyn Elizabeth Balasubramanian, Shankar 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. University of Cambridge 2017-12-05 Thesis Doctoral Doctor of Philosophy (PhD) en CHESS scholarship https://www.repository.cam.ac.uk/handle/1810/271807 https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/3bcf81d9-119a-4f75-921b-5be5dbf88c70/download 87eda9de84448d1f82354d60eee3eb5f https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/f3edca37-52f1-47b9-870e-8273b92af988/download 9eba70ff5b743a8324bf3c25df83ba06 https://www.rioxx.net/licenses/all-rights-reserved/ DNA modified bases epigenetics 5-hydroxymethyluracil 5-formyluracil 5-formylcytosine