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

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

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Base Sequence
  • Binding Sites
  • DNA Damage
  • DNA Repair*
  • DNA, Fungal / chemistry
  • DNA, Fungal / genetics*
  • DNA, Fungal / metabolism
  • DNA-Binding Proteins / chemistry
  • DNA-Binding Proteins / genetics*
  • DNA-Binding Proteins / metabolism
  • Gene Expression Regulation, Fungal*
  • Microscopy, Atomic Force
  • Microscopy, Fluorescence
  • Nucleic Acid Conformation
  • Protein Binding
  • Protein Conformation, alpha-Helical
  • Protein Conformation, beta-Strand
  • Protein Interaction Domains and Motifs
  • Quantum Dots / chemistry
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / metabolism
  • Saccharomyces cerevisiae / radiation effects*
  • Saccharomyces cerevisiae Proteins / chemistry
  • Saccharomyces cerevisiae Proteins / genetics*
  • Saccharomyces cerevisiae Proteins / metabolism
  • Sequence Deletion
  • Single Molecule Imaging
  • Ultraviolet Rays

Substances

  • DNA, Fungal
  • DNA-Binding Proteins
  • RAD23 protein, S cerevisiae
  • Rad4 protein, S cerevisiae
  • Saccharomyces cerevisiae Proteins