Single-molecule analysis reveals human UV-damaged DNA-binding protein (UV-DDB) dimerizes on DNA via multiple kinetic intermediates.
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.
Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213.
Harshad
H
Ghodke
Hong
H
Wang
Ching L
CL
Hsieh
Selamawit
S
Woldemeskel
Simon C
SC
Watkins
Vesna
V
Rapić-Otrin
Bennett
B
Van Houten
D019943:Amino Acid Substitution; D004247:DNA / Q000737:chemistry / Q000378:metabolism* / Q000528:radiation effects*; D004249:DNA Damage; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008856:Microscopy, Fluorescence; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D009994:Osmolar Concentration; D011487:Protein Conformation; D055503:Protein Multimerization; D011740:Pyrimidine Dimers / Q000378:metabolism / Q000528:radiation effects; D045663:Quantum Dots; D014466:Ultraviolet Rays / Q000009:adverse effects; D014983:Xeroderma Pigmentosum / Q000235:genetics / Q000378:metabolism
C091435:DDB1 protein, human; C117669:DDB2 protein, human; D004268:DNA-Binding Proteins; D011740:Pyrimidine Dimers; D004247:DNA
DNA damage recognition; DNA tightrope; human nucleotide excision repair; single-molecule tracking