Single-molecule analysis reveals human UV-damaged DNA-binding protein (UV-DDB) dimerizes on DNA via multiple kinetic intermediates

Proc Natl Acad Sci U S A. 2014 May 6;111(18):E1862-71. doi: 10.1073/pnas.1323856111. Epub 2014 Apr 23.

Abstract

How human DNA repair proteins survey the genome for UV-induced photoproducts remains a poorly understood aspect of the initial damage recognition step in nucleotide excision repair (NER). To understand this process, we performed single-molecule experiments, which revealed that the human UV-damaged DNA-binding protein (UV-DDB) performs a 3D search mechanism and displays a remarkable heterogeneity in the kinetics of damage recognition. Our results indicate that UV-DDB examines sites on DNA in discrete steps before forming long-lived, nonmotile UV-DDB dimers (DDB1-DDB2)2 at sites of damage. Analysis of the rates of dissociation for the transient binding molecules on both undamaged and damaged DNA show multiple dwell times over three orders of magnitude: 0.3-0.8, 8.1, and 113-126 s. These intermediate states are believed to represent discrete UV-DDB conformers on the trajectory to stable damage detection. DNA damage promoted the formation of highly stable dimers lasting for at least 15 min. The xeroderma pigmentosum group E (XP-E) causing K244E mutant of DDB2 found in patient XP82TO, supported UV-DDB dimerization but was found to slide on DNA and failed to stably engage lesions. These findings provide molecular insight into the loss of damage discrimination observed in this XP-E patient. This study proposes that UV-DDB recognizes lesions via multiple kinetic intermediates, through a conformational proofreading mechanism.

Keywords: DNA damage recognition; DNA tightrope; human nucleotide excision repair; single-molecule tracking.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Amino Acid Substitution
  • DNA / chemistry
  • DNA / metabolism*
  • DNA / radiation effects*
  • DNA Damage
  • DNA-Binding Proteins / chemistry*
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism*
  • Humans
  • Kinetics
  • Microscopy, Fluorescence
  • Models, Molecular
  • Mutagenesis, Site-Directed
  • Nucleic Acid Conformation
  • Osmolar Concentration
  • Protein Conformation
  • Protein Multimerization
  • Pyrimidine Dimers / metabolism
  • Pyrimidine Dimers / radiation effects
  • Quantum Dots
  • Ultraviolet Rays / adverse effects
  • Xeroderma Pigmentosum / genetics
  • Xeroderma Pigmentosum / metabolism

Substances

  • DDB1 protein, human
  • DDB2 protein, human
  • DNA-Binding Proteins
  • Pyrimidine Dimers
  • DNA