Journal article
Single-Molecule Imaging Reveals that Rad4 Employs a Dynamic DNA Damage Recognition Process
Molecular cell, Vol.64(2), pp.376-387
10/20/2016
Handle:
https://hdl.handle.net/2376/111064
PMCID: PMC5123691
PMID: 27720644
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.
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•Rad4-Rad23 exhibits lesion-dependent random and constrained 1D diffusion on DNA•Initial damage detection and DNA bending can be achieved without BHD3•Mutant with increased constrained motion in vitro allows efficient repair in vivo
Kong et al. demonstrate that Rad4-Rad23, the damage sensor in yeast nucleotide excision repair, exhibits lesion-dependent static binding or one-dimensional diffusion on DNA, the latter displaying random or constrained diffusive behavior. Such constrained motion is shown to be biologically relevant for in vivo repair and hints at a broader “recognition-at-a-distance” model.
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Details
- Title
- Single-Molecule Imaging Reveals that Rad4 Employs a Dynamic DNA Damage Recognition Process
- Creators
- Muwen Kong - Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USALili Liu - Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USAXuejing Chen - Department of Chemistry, University of Illinois at Chicago, Chicago, IL 60607, USAKatherine I Driscoll - Department of Physics and Astronomy, University of South Carolina, Columbia, SC 29208, USAPeng Mao - School of Molecular Biosciences, Washington State University, Pullman, WA 99164, USAStefanie Böhm - Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USANeil M Kad - School of Biosciences, University of Kent, Canterbury, Kent CT2 7NJ, UKSimon C Watkins - Center for Biologic Imaging, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USAKara A Bernstein - Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USAJohn J Wyrick - School of Molecular Biosciences, Washington State University, Pullman, WA 99164, USAJung-Hyun Min - Department of Chemistry, University of Illinois at Chicago, Chicago, IL 60607, USABennett Van Houten - Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA
- Publication Details
- Molecular cell, Vol.64(2), pp.376-387
- Academic Unit
- Molecular Biosciences, School of
- Publisher
- Elsevier Inc
- Identifiers
- 99900547384901842
- Language
- English
- Resource Type
- Journal article