title: Single-Molecule Imaging Reveals that Rad4 Employs a Dynamic DNA Damage Recognition Process.
abstract: 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.
authors.affiliation: Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA; University of Pittsburgh Cancer Institute, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA.
authors.forename: Muwen
authors.initials: M
authors.lastname: Kong
authors.affiliation: Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA; University of Pittsburgh Cancer Institute, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA.
authors.forename: Lili
authors.initials: L
authors.lastname: Liu
authors.affiliation: Department of Chemistry, University of Illinois at Chicago, Chicago, IL 60607, USA.
authors.forename: Xuejing
authors.initials: X
authors.lastname: Chen
authors.affiliation: Department of Physics and Astronomy, University of South Carolina, Columbia, SC 29208, USA.
authors.forename: Katherine I
authors.initials: KI
authors.lastname: Driscoll
authors.affiliation: School of Molecular Biosciences, Washington State University, Pullman, WA 99164, USA.
authors.forename: Peng
authors.initials: P
authors.lastname: Mao
authors.affiliation: Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA; University of Pittsburgh Cancer Institute, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA.
authors.forename: Stefanie
authors.initials: S
authors.lastname: Böhm
authors.affiliation: School of Biosciences, University of Kent, Canterbury, Kent CT2 7NJ, UK.
authors.forename: Neil M
authors.initials: NM
authors.lastname: Kad
authors.affiliation: Center for Biologic Imaging, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA.
authors.forename: Simon C
authors.initials: SC
authors.lastname: Watkins
authors.affiliation: Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA; University of Pittsburgh Cancer Institute, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA.
authors.forename: Kara A
authors.initials: KA
authors.lastname: Bernstein
authors.affiliation: School of Molecular Biosciences, Washington State University, Pullman, WA 99164, USA.
authors.forename: John J
authors.initials: JJ
authors.lastname: Wyrick
authors.affiliation: Department of Chemistry, University of Illinois at Chicago, Chicago, IL 60607, USA. Electronic address: jhmin@uic.edu.
authors.forename: Jung-Hyun
authors.initials: JH
authors.lastname: Min
authors.affiliation: Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA; University of Pittsburgh Cancer Institute, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA. Electronic address: vanhoutenb@upmc.edu.
authors.forename: Bennett
authors.initials: B
authors.lastname: Van Houten
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D004249:DNA Damage; D004260:DNA Repair; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D018625:Microscopy, Atomic Force; D008856:Microscopy, Fluorescence; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D000072756:Protein Conformation, alpha-Helical; D000072757:Protein Conformation, beta-Strand; D054730:Protein Interaction Domains and Motifs; D045663:Quantum Dots / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism / Q000528:radiation effects*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D017384:Sequence Deletion; D000072760:Single Molecule Imaging; D014466:Ultraviolet Rays
chemical_list: D004271:DNA, Fungal; D004268:DNA-Binding Proteins; C490846:RAD23 protein, S cerevisiae; C493797:Rad4 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins
keywords: DNA tightrope assay; Rad23; Rad4; XPC; dynamic DNA damage recognition; nucleotide excision repair; quantum dots; single particle tracking; xeroderma pigmentosum