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Title: Methods to probe the function of modified bases in DNA
Author: Hardisty, Robyn Elizabeth
ISNI:       0000 0004 7225 9345
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2017
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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.
Supervisor: Balasubramanian, Shankar Sponsor: CHESS
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
Keywords: DNA ; modified bases ; epigenetics ; 5-hydroxymethyluracil ; 5-formyluracil ; 5-formylcytosine