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Title: Real time single molecule investigation of the dynamics of nucleotide excision repair proteins on DNA tightropes
Author: Hughes, Craig David
ISNI:       0000 0004 5358 672X
Awarding Body: University of Essex
Current Institution: University of Essex
Date of Award: 2014
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Through the use of a biochemical technique that allows single strands of DNA to be visualized, the dynamics of nucleotide excision repair proteins was studied at the single molecule level. This DNA tightrope technique has allowed for the interaction of proteins with double stranded, single stranded, crowded and damaged DNA to be observed. Bacterial nucleotide excision repair proteins UvrC and UvrBC were loaded onto undamaged DNA to understand how UvrC locates the UvrB-DNA pre-incision complex. It was obseNed that UvrC facilitates UvrB binding to DNA in the absence of UvrA and as this UvrBC complex was abundant, indicates that UvrC is most likely present in an UvrBC complex in vivo. Mutant studies demonstrated that UvrBC binds to DNA through UvrB's DNA binding domain, signifying that the UvrBC complex forms in order to help UvrC locate an UvrB pre-incision DNA damage complex and to stop unchecked endonuclease activity. It was proposed that this UvrBC complex may also benefit UvrC in maneuvering around obstructions on the DNA. Within a cell DNA will always be involved in a multitude of differentreactions concurrently, therefore obseNations on how NER proteins behaved on crowded DNA was studied. These results demonstrated that both UvrBC and UvrC have difficulties overcoming protein obstructions. This suggests that the main mechanism of movement of UvrC and UvrBC on the DNA is a one dimensional search, with the possible capability of switching to a 3D search. The eukaryotic NER protein XPD was also imaged, demonstrating a single stranded DNA binding preference over that of double stranded DNA. The XPD protein was also obseNed to have an absolute requirement for A TP in order to have motion on the DNA. Once damage was introduced into the DNA it was obseNed that UvrB facilitates UvrC binding to damaged DNA, through the UvrB-DNA binding domain. This previously unreported damage detection mechanism of UvrBC originates from UvrB's ~-hairpin motif's interaction with DNA. It was therefore suggested that UvrBC's damage .detection is a redundancy system in the NER pathway.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available