Muwen Kong^1,3, Lili Liu^1,3, Xuejing Chen⁴, Katherine I. Driscoll⁵, Peng Mao⁶, Stefanie Böhm^2,3, Neil M. Kad⁷, Simon C. Watkins⁸, Kara A. Bernstein^2,3, John J. Wyrick⁶, Jung-Hyun Min^4,*, Bennett Van Houten<sup>1,3,9,*

1</sup> Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA
² Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA
³ University of Pittsburgh Cancer Institute, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA
⁴ Department of Chemistry, University of Illinois at Chicago, Chicago, IL 60607, USA
⁵ Department of Physics and Astronomy, University of South Carolina, Columbia, SC 29208, USA
⁶ School of Molecular Biosciences, Washington State University, Pullman, WA 99164, USA
⁷ School of Biosciences, University of Kent, Canterbury, Kent CT2 7NJ, UK
⁸ Center for Biologic Imaging, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA
⁹ Lead Contact

^*Correspondence : Jung-Hyun Min, Bennett Van Houten

Summary
Nucleotide excision repair (NER) is an evolutionarily conserved mechanism that processes helix-destabilizing and/or -distorting DNA lesions, such as UV-induced photoproducts. Here, we investigate the dynamic protein-DNA interactions during the damage recognition step using single-molecule fluorescence microscopy. Quantum dot-labeled Rad4-Rad23 (yeast XPC-RAD23B ortholog) forms non-motile complexes or conducts a one-dimensional search via either random diffusion or constrained motion. Atomic force microcopy analysis of Rad4 with the β-hairpin domain 3 (BHD3) deleted reveals that this motif is non-essential for damage-specific binding and DNA bending. Furthermore, we find that deletion of seven residues in the tip of β-hairpin in BHD3 increases 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.

Keywords: Rad4, Rad23, XPC, nucleotide excision repair, xeroderma pigmentosum, single particle tracking, dynamic DNA damage recognition, DNA tightrope assay, quantum dots