{"id":{"repo_id":"cambridge","oai_identifier":"oai:www.repository.cam.ac.uk:1810/271807"},"canonical_url":"https://search.dev.ndltd.org/etd/cambridge/oai:www.repository.cam.ac.uk:1810/271807","repository":{"repo_id":"cambridge","name":"Cambridge University","base_url":"https://api.repository.cam.ac.uk/server/oai/request"},"display":{"title":"Methods to Probe the Function of Modified Bases in DNA","abstract":"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.","abstract_html":"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.","abstract_has_math":false,"creators":["Hardisty, Robyn Elizabeth"],"institution":"University of Cambridge","degree_name":"Doctor of Philosophy (PhD)","degree_level":"Doctoral","degree_discipline":null,"degree_department":null,"school":null,"contributors":[],"advisors":["Balasubramanian, Shankar"],"committee_chairs":[],"committee_members":[],"year":2017,"date_issued":"2017-12-05","date_published":"2017-12-05","updated_at":"2026-07-22T22:24:04Z","subjects":["DNA","modified bases","epigenetics","5-hydroxymethyluracil","5-formyluracil","5-formylcytosine"],"languages":["en"],"rights":[],"rights_urls":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/3bcf81d9-119a-4f75-921b-5be5dbf88c70/download","https://www.rioxx.net/licenses/all-rights-reserved/"],"identifier_entries":[]},"links":{"outbound_url":"https://doi.org/10.17863/CAM.18804","outbound_label":"DOI","outbound_source":"dc:identifier.doi"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.advisor","label":"Advisor","values":["Balasubramanian, Shankar"]},{"key":"dc:contributor.sponsor","label":"Sponsor","values":["CHESS scholarship"]},{"key":"dc:creator","label":"Author","values":["Hardisty, Robyn Elizabeth"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.issued","label":"Date","values":["2017-12-05"]},{"key":"dc:publisher.institution","label":"Dc Publisher Institution","values":["University of Cambridge"]},{"key":"dc:relation.isreferencedby.uri","label":"Dc Relation Isreferencedby URI","values":["https://www.repository.cam.ac.uk/handle/1810/271807"]},{"key":"dc:type","label":"Dc Type","values":["Thesis"]},{"key":"dc:type.qualificationlevel","label":"Dc Type Qualificationlevel","values":["Doctoral"]},{"key":"dc:type.qualificationname","label":"Dc Type Qualificationname","values":["Doctor of Philosophy (PhD)"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["DNA","modified bases","epigenetics","5-hydroxymethyluracil","5-formyluracil","5-formylcytosine"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["en"]},{"key":"dc:rights","label":"Dc Rights","values":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/3bcf81d9-119a-4f75-921b-5be5dbf88c70/download","https://www.rioxx.net/licenses/all-rights-reserved/"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.doi","label":"DOI","values":["10.17863/CAM.18804"]},{"key":"dc:identifier.uri","label":"Identifier URI","values":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/f3edca37-52f1-47b9-870e-8273b92af988/download"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["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. 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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. 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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. 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