{"id":{"repo_id":"uiuc","oai_identifier":"oai:www.ideals.illinois.edu:2142/124689"},"canonical_url":"https://search.dev.ndltd.org/etd/uiuc/oai:www.ideals.illinois.edu:2142/124689","repository":{"repo_id":"uiuc","name":"University of Illinois - Urbana-Champaign","base_url":"https://www.ideals.illinois.edu/oai-pmh"},"display":{"title":"Expeditious detection of DNA damage: Probing the sequence-sensing and damage-sensing mechanism(s) of the XPD helicase","abstract":"Submission published under a 24 month embargo labeled 'Closed Access', the embargo will last until 2026-05-01","abstract_html":"Submission published under a 24 month embargo labeled 'Closed Access', the embargo will last until 2026-05-01","abstract_has_math":false,"creators":["Troitskaia, Alice"],"institution":"University of Illinois at Urbana-Champaign","degree_name":"Ph.D.","degree_level":"Dissertation","degree_discipline":"Biophysics & Quant Biology","degree_department":null,"school":null,"contributors":["Chemla, Yann","Selvin, Paul R","Luthey-Schulten, Zaida A","Kim, Sangjin"],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2024,"date_issued":"2024-05","date_published":"2024-05","updated_at":"2026-07-22T22:25:02Z","subjects":["Dna Helicases","Dna Repair","Dna Damage Sensing","Nucleotide Excision Repair","Single Molecule","Optical Trapping"],"languages":["en","eng"],"rights":["Copyright 2024 Alice Troitskaia"],"rights_urls":[],"identifier_entries":[]},"links":{"outbound_url":"https://hdl.handle.net/2142/124689","outbound_label":"Handle","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["Chemla, Yann","Selvin, Paul R","Luthey-Schulten, Zaida A","Kim, Sangjin"]},{"key":"dc:creator","label":"Author","values":["Troitskaia, Alice"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date","label":"Dc Date","values":["2024-05","2024-04-26"]},{"key":"dc:type","label":"Dc Type","values":["text"]},{"key":"thesis:degree_discipline","label":"Discipline","values":["Biophysics & Quant Biology"]},{"key":"thesis:degree_level","label":"Degree Level","values":["Dissertation"]},{"key":"thesis:degree_name","label":"Degree Name","values":["Ph.D."]},{"key":"thesis:institution_name","label":"Thesis Institution Name","values":["University of Illinois at Urbana-Champaign"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["Dna Helicases","Dna Repair","Dna Damage Sensing","Nucleotide Excision Repair","Single Molecule","Optical Trapping"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["en","eng"]},{"key":"dc:rights","label":"Dc Rights","values":["Copyright 2024 Alice Troitskaia"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["https://hdl.handle.net/2142/124689"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description","label":"Description","values":["Submission published under a 24 month embargo labeled 'Closed Access', the embargo will last until 2026-05-01","The student, Alice Troitskaia, accepted the attached license on 2024-04-23 at 11:23.","The student, Alice Troitskaia, submitted this Dissertation for approval on 2024-04-23 at 13:03.","This Dissertation was approved for publication on 2024-04-26 at 08:43.","DSpace SAF Submission Ingestion Package generated from Vireo submission #20568 on 2024-09-16 at 00:50:06","Practically any process involving DNA in living cells relies on helicases, enzymes which unwind double-stranded DNA. Some helicases may play different roles in different processes. XPD (Xeroderma pigmentosum group D), a member of the transcription factor IIH complex, plays a structural role in transcription, but its helicase and ATPase activity are critical to nucleotide excision repair. In addition to unwinding the duplex around a damage site, some variants of XPD have been implicated in damage verification. XPD’s close contact with DNA puts it in an ideal position to interrogate DNA for damage; prior work has shown that it is very sensitive to DNA energetics, and residues have been identified which appear to be important for damage detection. However, there is still a lack of consensus as to how XPD responds to various kinds of lesions or modifications, and how the response depends on the strand they reside on. The possibility of different mechanisms to sense different types of damage has been proposed. Determining details of such mechanisms is challenging as there is limited information on the dynamics of XPD during damage detection. Using a single-molecule optical trapping assay, we measure the unwinding dynamics of XPD from the archaeon Ferroplasma acidarmanus (FacXPD) on sequences with specific energetic properties and incorporating different types of DNA damage. We first build on prior work from our group on XPD’s sensitivity to DNA energetics, by designing and synthesizing energetically uniform hairpin constructs and measuring XPD unwinding activity on these substrates. We find that single XPD exhibits a wide variety of behaviours on these energetically uniform hairpins. Interestingly, despite the energetic uniformity, XPD appears to spend more time on certain regions of the sequence, suggesting subtle sequence sensing. The specific motifs resulting in increased dwells have so far been elusive; however, the patterns of increased dwell time clearly track with sequence. As these sequences minimize the effects of DNA energetics on XPD dynamics, we then use them as a platform for measuring the effects of damage on XPD unwinding activity. Different modifications are incorporated into DNA substrates at well-defined positions on either strand. Measurements of unwinding activity on these substrates show that FacXPD responds differently to different lesions, apparently implicating different parts of the protein. Our results support the existence of multiple mechanisms for damage sensing, with multiple mechanisms sometimes being invoked even for the same modification. Detection appears to take place on the translocated strand only. Surprisingly, we find that a mutant characterized as damage-sensing by a different study responds to damage in a similar manner to wild-type XPD in our assay. This result suggests that damage sensing in that region of the protein is more complex that the interaction of that single residue with a DNA damage site. Ongoing work suggests that interactions between multiple XPDs may change the nature of the helicase’s interaction with damage, as separate work shows that XPD unwinding activity on undamaged DNA is altered by protein-protein interactions between XPD and a cognate single-strand DNA binding protein."]},{"key":"dc:format","label":"Dc Format","values":["application/pdf"]},{"key":"dc:title","label":"Title","values":["Expeditious detection of DNA damage: Probing the sequence-sensing and damage-sensing mechanism(s) of the XPD helicase"]}]}],"canonical_facts":{"dc:contributor":["Chemla, Yann","Selvin, Paul R","Luthey-Schulten, Zaida A","Kim, Sangjin"],"dc:creator":["Troitskaia, Alice"],"dc:date":["2024-05","2024-04-26"],"dc:description":["Submission published under a 24 month embargo labeled 'Closed Access', the embargo will last until 2026-05-01","The student, Alice Troitskaia, accepted the attached license on 2024-04-23 at 11:23.","The student, Alice Troitskaia, submitted this Dissertation for approval on 2024-04-23 at 13:03.","This Dissertation was approved for publication on 2024-04-26 at 08:43.","DSpace SAF Submission Ingestion Package generated from Vireo submission #20568 on 2024-09-16 at 00:50:06","Practically any process involving DNA in living cells relies on helicases, enzymes which unwind double-stranded DNA. Some helicases may play different roles in different processes. XPD (Xeroderma pigmentosum group D), a member of the transcription factor IIH complex, plays a structural role in transcription, but its helicase and ATPase activity are critical to nucleotide excision repair. In addition to unwinding the duplex around a damage site, some variants of XPD have been implicated in damage verification. XPD’s close contact with DNA puts it in an ideal position to interrogate DNA for damage; prior work has shown that it is very sensitive to DNA energetics, and residues have been identified which appear to be important for damage detection. However, there is still a lack of consensus as to how XPD responds to various kinds of lesions or modifications, and how the response depends on the strand they reside on. The possibility of different mechanisms to sense different types of damage has been proposed. Determining details of such mechanisms is challenging as there is limited information on the dynamics of XPD during damage detection. Using a single-molecule optical trapping assay, we measure the unwinding dynamics of XPD from the archaeon Ferroplasma acidarmanus (FacXPD) on sequences with specific energetic properties and incorporating different types of DNA damage. We first build on prior work from our group on XPD’s sensitivity to DNA energetics, by designing and synthesizing energetically uniform hairpin constructs and measuring XPD unwinding activity on these substrates. We find that single XPD exhibits a wide variety of behaviours on these energetically uniform hairpins. Interestingly, despite the energetic uniformity, XPD appears to spend more time on certain regions of the sequence, suggesting subtle sequence sensing. The specific motifs resulting in increased dwells have so far been elusive; however, the patterns of increased dwell time clearly track with sequence. As these sequences minimize the effects of DNA energetics on XPD dynamics, we then use them as a platform for measuring the effects of damage on XPD unwinding activity. Different modifications are incorporated into DNA substrates at well-defined positions on either strand. Measurements of unwinding activity on these substrates show that FacXPD responds differently to different lesions, apparently implicating different parts of the protein. Our results support the existence of multiple mechanisms for damage sensing, with multiple mechanisms sometimes being invoked even for the same modification. Detection appears to take place on the translocated strand only. Surprisingly, we find that a mutant characterized as damage-sensing by a different study responds to damage in a similar manner to wild-type XPD in our assay. This result suggests that damage sensing in that region of the protein is more complex that the interaction of that single residue with a DNA damage site. Ongoing work suggests that interactions between multiple XPDs may change the nature of the helicase’s interaction with damage, as separate work shows that XPD unwinding activity on undamaged DNA is altered by protein-protein interactions between XPD and a cognate single-strand DNA binding protein."],"dc:format":["application/pdf"],"dc:identifier":["https://hdl.handle.net/2142/124689"],"dc:language":["en","eng"],"dc:rights":["Copyright 2024 Alice Troitskaia"],"dc:subject":["Dna Helicases","Dna Repair","Dna Damage Sensing","Nucleotide Excision Repair","Single Molecule","Optical Trapping"],"dc:title":["Expeditious detection of DNA damage: Probing the sequence-sensing and damage-sensing mechanism(s) of the XPD helicase"],"dc:type":["text"],"thesis:degree_discipline":["Biophysics & Quant Biology"],"thesis:degree_level":["Dissertation"],"thesis:degree_name":["Ph.D."],"thesis:institution_name":["University of Illinois at Urbana-Champaign"]},"updated_at":"2026-07-22T22:25:02Z"}