title: Novel method for site-specific induction of oxidative DNA damage reveals differences in recruitment of repair proteins to heterochromatin and euchromatin.
abstract: 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.
authors.affiliation: Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA, University of Pittsburgh Cancer Institute, University of Pittsburgh School of Medicine, 5117 Centre Avenue, Pittsburgh, PA 15213, USA, School of Medicine, Tsinghua University, No.1 Tsinghua Yuan, Haidian District, Beijing 100084, People's Republic of China, Department of Pharmacology & Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA, Department of Human Genetics, University of Pittsburgh Graduate School of Public Health, Pittsburgh, PA 15213, USA, Department of Chemistry and Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA 15213, USA and Division of Dynamic Proteome, Institute of Development, Aging, and Cancer, Tohoku University, Seiryomachi 4-1, Sendai 980-8575, Japan.
authors.forename: Li
authors.initials: L
authors.lastname: Lan
authors.forename: Satoshi
authors.initials: S
authors.lastname: Nakajima
authors.forename: Leizhen
authors.initials: L
authors.lastname: Wei
authors.forename: Luxi
authors.initials: L
authors.lastname: Sun
authors.forename: Ching-Lung
authors.initials: CL
authors.lastname: Hsieh
authors.forename: Robert W
authors.initials: RW
authors.lastname: Sobol
authors.forename: Marcel
authors.initials: M
authors.lastname: Bruchez
authors.forename: Bennett
authors.initials: B
authors.lastname: Van Houten
authors.forename: Akira
authors.initials: A
authors.lastname: Yasui
authors.forename: Arthur S
authors.initials: AS
authors.lastname: Levine
mesh_terms: D002460:Cell Line; D002843:Chromatin / Q000378:metabolism; D004249:DNA Damage; D045647:DNA Glycosylases / Q000378:metabolism; D019951:DNA Polymerase beta / Q000378:metabolism; D004260:DNA Repair; D045643:DNA Repair Enzymes / Q000378:metabolism*; D022041:Euchromatin / Q000201:enzymology / Q000378:metabolism*; D045585:Flap Endonucleases / Q000378:metabolism; D016678:Genome; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000528:radiation effects; D006570:Heterochromatin / Q000201:enzymology / Q000378:metabolism*; D006801:Humans; D007834:Lasers; D010084:Oxidation-Reduction; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000032:analysis; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism; D020218:Response Elements; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism
chemical_list: D002843:Chromatin; D022041:Euchromatin; D006570:Heterochromatin; D018809:Proliferating Cell Nuclear Antigen; D017382:Reactive Oxygen Species; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D015534:Trans-Activators; C508771:killer red protein, Anthomedusae; C034811:tetracycline resistance-encoding transposon repressor protein; D049452:Green Fluorescent Proteins; C499320:PARP1 protein, human; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases; D019951:DNA Polymerase beta; D045585:Flap Endonucleases; D045647:DNA Glycosylases; D045643:DNA Repair Enzymes