Novel method for site-specific induction of oxidative DNA damage reveals differences in recruitment of repair proteins to heterochromatin and euchromatin

Nucleic Acids Res. 2014 Feb;42(4):2330-45. doi: 10.1093/nar/gkt1233. Epub 2013 Nov 29.


Reactive oxygen species (ROS)-induced DNA damage is repaired by the base excision repair pathway. However, the effect of chromatin structure on BER protein recruitment to DNA damage sites in living cells is poorly understood. To address this problem, we developed a method to specifically produce ROS-induced DNA damage by fusing KillerRed (KR), a light-stimulated ROS-inducer, to a tet-repressor (tetR-KR) or a transcription activator (TA-KR). TetR-KR or TA-KR, bound to a TRE cassette (∼ 90 kb) integrated at a defined genomic locus in U2OS cells, was used to induce ROS damage in hetero- or euchromatin, respectively. We found that DNA glycosylases were efficiently recruited to DNA damage in heterochromatin, as well as in euchromatin. PARP1 was recruited to DNA damage within condensed chromatin more efficiently than in active chromatin. In contrast, recruitment of FEN1 was highly enriched at sites of DNA damage within active chromatin in a PCNA- and transcription activation-dependent manner. These results indicate that oxidative DNA damage is differentially processed within hetero or euchromatin.

Publication types

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

MeSH terms

  • Cell Line
  • Chromatin / metabolism
  • DNA Damage*
  • DNA Glycosylases / metabolism
  • DNA Polymerase beta / metabolism
  • DNA Repair Enzymes / metabolism*
  • DNA Repair*
  • Euchromatin / enzymology
  • Euchromatin / metabolism*
  • Flap Endonucleases / metabolism
  • Genome
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / radiation effects
  • Heterochromatin / enzymology
  • Heterochromatin / metabolism*
  • Humans
  • Lasers
  • Oxidation-Reduction
  • Poly (ADP-Ribose) Polymerase-1
  • Poly(ADP-ribose) Polymerases / metabolism
  • Proliferating Cell Nuclear Antigen / metabolism
  • Reactive Oxygen Species / metabolism
  • Recombinant Fusion Proteins / analysis
  • Repressor Proteins / genetics
  • Repressor Proteins / metabolism
  • Response Elements
  • Trans-Activators / genetics
  • Trans-Activators / metabolism


  • Chromatin
  • Euchromatin
  • Heterochromatin
  • Proliferating Cell Nuclear Antigen
  • Reactive Oxygen Species
  • Recombinant Fusion Proteins
  • Repressor Proteins
  • Trans-Activators
  • killer red protein, Anthomedusae
  • tetracycline resistance-encoding transposon repressor protein
  • Green Fluorescent Proteins
  • PARP1 protein, human
  • Poly (ADP-Ribose) Polymerase-1
  • Poly(ADP-ribose) Polymerases
  • DNA Polymerase beta
  • Flap Endonucleases
  • DNA Glycosylases
  • DNA Repair Enzymes