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Combining Single Molecule and Bulk Phase Approaches to Investigate Damage Detection by Nucleotide Excision Repair Proteins

Barnett, Jamie (2019) Combining Single Molecule and Bulk Phase Approaches to Investigate Damage Detection by Nucleotide Excision Repair Proteins. Doctor of Philosophy (PhD) thesis, University of Kent,. (KAR id:79545)


DNA repair is crucial for the survival of all organisms. However, cellular DNA repair pathways are complex and many of the molecular details remain elusive. Understanding how DNA is repaired is paramount, as aberrant repair is implicated in numerous diseases that lead to premature ageing and increased cancer risk. Nucleotide excision repair (NER) is found across all kingdoms of life and protects the genome following exposure to diverse types of DNA damage, including UV light and chemotherapeutics. In this thesis, a combination of single molecular fluorescence and bulk biochemical techniques are used to investigate how NER proteins find damage in the genome. Individual molecules of DNA repair proteins are visualised interacting with the DNA using fluorescent tags. We show both DNA damage and nucleotide cofactors affect the attached lifetime of a prokaryotic DNA damage detecting protein (UvrA) to DNA. Based on these data and supporting biochemical ATPase experiments we devise a hypothesis where UvrA sequentially hydrolyses nucleotide in each ATPase site and reconcile this with UvrA's role in DNA repair. We also investigate two recent cryo-EM structures of the major transcription factor TFIH. Two previously identified 'structural' subunits (p44/p62) are show to make extensive contacts with a damage verifying helicase, XPD. We show these subunits stimulate XPD's ATPase on damaged DNA. In addition, we show p44/p62 form an independent complex that is capable of binding to DNA and discriminating damage. This suggests p44/p62 is a novel DNA-binding entity in the TFIH complex that may have a role in the detection or verification of DNA damage. The results in this have relevance for understanding cancer and ageing, as well as answering controversial questions in the field and contributing to our understanding of how DNA is repaired.

Item Type: Thesis (Doctor of Philosophy (PhD))
Thesis advisor: Kad, Neil
Uncontrolled keywords: Biochemistry, Single-molecule, Kinetics, DNA repair
Subjects: Q Science > QP Physiology (Living systems) > QP517 Biochemistry
Divisions: Divisions > Division of Natural Sciences > Biosciences
Funders: Organisations -1 not found.
SWORD Depositor: System Moodle
Depositing User: System Moodle
Date Deposited: 13 Jan 2020 11:13 UTC
Last Modified: 08 Dec 2022 23:51 UTC
Resource URI: (The current URI for this page, for reference purposes)

University of Kent Author Information

Barnett, Jamie.

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