Dissertation/Thesis Abstract

Single-Molecule Studies of Rad4-Rad23 Reveal a Dynamic DNA Damage Recognition Process
by Kong, Muwen, Ph.D., University of Pittsburgh, 2017, 205; 10692541
Abstract (Summary)

Nucleotide excision repair (NER) is an evolutionarily conserved mechanism that processes helixdestabilizing 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.

Indexing (document details)
Advisor: Houten, Bennett Van
Commitee: Bruchez, Marcel, Kad, Neil, Opresko, Patricia
School: University of Pittsburgh
Department: Molecular Biophysics and Structural Biology
School Location: United States -- Pennsylvania
Source: DAI-B 79/04(E), Dissertation Abstracts International
Subjects: Molecular biology, Biochemistry, Biophysics
Keywords: XPC-RAD23B, atomic force microscopy, nucleotide excision repair, single-molecule fluirescence microscopy, subdiffusion
Publication Number: 10692541
ISBN: 9780355410143
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