Kong, Muwen
(2017)
SINGLE-MOLECULE STUDIES OF RAD4-RAD23 REVEAL A DYNAMIC DNA DAMAGE RECOGNITION PROCESS.
Doctoral Dissertation, University of Pittsburgh.
(Unpublished)
Abstract
Nucleotide excision repair (NER) is an evolutionarily conserved mechanism that processes helix-destabilizing and/or -distorting DNA lesions, such as UV-induced photoproducts. As the first step towards productive repair, the human NER damage sensor XPC-RAD23B needs to efficiently locate sites of damage among billons of base pairs of undamaged DNA. In this dissertation, we investigated the dynamic protein-DNA interactions during the damage recognition step using a combination of fluorescence-based single-molecule DNA tightrope assays, atomic force microscopy, as well as cell survival and in vivo repair kinetics assays. We observed that quantum dot-labeled Rad4-Rad23, the yeast homolog of human XPC-RAD23B, formed nonmotile complexes on DNA or conducted a one-dimensional search via either random diffusion or constrained motion along DNA. Using atomic force microscopy, we studied binding of Rad4 lacking the β-hairpin domain 3 (BHD3) to damage-containing DNA and found that this structural motif is non-essential for damage-specific binding or DNA bending. Furthermore, we demonstrated that deletion of seven residues in the tip of β-hairpin in BHD3 increased Rad4-Rad23 constrained motion at the expense of stable binding at sites of DNA lesions, without diminishing cellular UV resistance or photoproduct repair in vivo. These results suggest a distinct intermediate in the damage recognition process during NER, allowing dynamic DNA damage detection at a distance. Finally, we explore existing physical models and examples of subdiffusive motion, and discuss a model in which constrained motion by Rad4-Rad23 on DNA may be driven by conformational changes of the protein.
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Details
| Item Type: |
University of Pittsburgh ETD
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| Status: |
Unpublished |
| Creators/Authors: |
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| ETD Committee: |
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| Date: |
8 August 2017 |
| Date Type: |
Publication |
| Defense Date: |
30 June 2017 |
| Approval Date: |
8 August 2017 |
| Submission Date: |
4 August 2017 |
| Access Restriction: |
No restriction; Release the ETD for access worldwide immediately. |
| Number of Pages: |
200 |
| Institution: |
University of Pittsburgh |
| Schools and Programs: |
School of Medicine > Molecular Biophysics and Structural Biology |
| Degree: |
PhD - Doctor of Philosophy |
| Thesis Type: |
Doctoral Dissertation |
| Refereed: |
Yes |
| Uncontrolled Keywords: |
Rad4, Rad23, single-molecule, fluorescence microscopy, atomic force microscopy, nucleotide excision repair, subdiffusion, DNA tightrope assay, single particle tracking, xeroderma pigmentosum, XPC |
| Date Deposited: |
08 Aug 2017 15:46 |
| Last Modified: |
08 Aug 2017 15:46 |
| URI: |
http://d-scholarship.pitt.edu/id/eprint/32997 |
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