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Mol Cell. 2016 Oct 20;64(2):376-387. doi: 10.1016/j.molcel.2016.09.005. Epub 2016 Oct 6.

Single-Molecule Imaging Reveals that Rad4 Employs a Dynamic DNA Damage Recognition Process.

Author information

1
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.
2
Department of Chemistry, University of Illinois at Chicago, Chicago, IL 60607, USA.
3
Department of Physics and Astronomy, University of South Carolina, Columbia, SC 29208, USA.
4
School of Molecular Biosciences, Washington State University, Pullman, WA 99164, USA.
5
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.
6
School of Biosciences, University of Kent, Canterbury, Kent CT2 7NJ, UK.
7
Center for Biologic Imaging, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA.
8
Department of Chemistry, University of Illinois at Chicago, Chicago, IL 60607, USA. Electronic address: jhmin@uic.edu.
9
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.

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.

KEYWORDS:

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

PMID:
27720644
PMCID:
PMC5123691
DOI:
10.1016/j.molcel.2016.09.005
[Indexed for MEDLINE]
Free PMC Article

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