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Kong, Muwen (2017) SINGLE-MOLECULE STUDIES OF RAD4-RAD23 REVEAL A DYNAMIC DNA DAMAGE RECOGNITION PROCESS. Doctoral Dissertation, University of Pittsburgh. (Unpublished)

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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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Item Type: University of Pittsburgh ETD
Status: Unpublished
CreatorsEmailPitt UsernameORCID
Kong, Muwenmuwenkon@pitt.edumuwenkon0000-0002-7845-1165
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee ChairRomero,
Thesis AdvisorVan Houten,
Committee MemberBruchez,
Committee MemberPatricia,
Committee MemberNeil,
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


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