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Methods Mol Biol. 2017;1587:139-146. doi: 10.1007/978-1-4939-6892-3_14.

Induction of Site-Specific Oxidative Damage at Telomeres by Killerred-Fused Shelretin Proteins.

Tan R1,2,3, Lan L4,5.

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University of Pittsburgh Cancer Institute, 5117 Centre Avenue, Pittsburgh, PA, 15213, USA.
Xiangya Hospital, Central South University, 87 Xiangya Road, Changsha, Hunan, 410008, China.
University of Pittsburgh School of Medicine, 5117 Centre Avenue, Pittsburgh, PA, 15213, USA.
University of Pittsburgh Cancer Institute, 5117 Centre Avenue, Pittsburgh, PA, 15213, USA.
Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Bridgeside Point II, 450 Technology Drive, Pittsburgh, PA, 15219, USA.


Chronic oxidative stress is the major endogenous metabolic stress and contributes directly to telomere shortening and senescence. Understanding the dysfunction of telomeres under oxidative stress will greatly facilitate healthy aging and advance the treatment of aging-related diseases. Here, we describe the KR-TEL (KillerRed induced DNA damage at telomeres) system that induces site-specific oxidative damage at telomeres. We have developed the KR-TEL system by fusing killerred with the shelterin component TRF1 (KR-TRF1) or other shelterin proteins. Killerred (KR), an engineered red fluorescent chromophore, is capable of generating site-specific superoxide upon green light activation (550-580 nm). When KR-TRF1 expressing cells are exposed to green or laser light at defined wavelength to activate KR, localized oxidative DNA damage will be induced at telomeres. KR-induced oxidative DNA damage shows a high degree of resemblance to the complex spectrum of DNA damage induced by radiation in terms of the ratios of oxidized bases and DNA strand breaks. Unlike current oxidation-inducing methods (e.g., IR, chemical, and toxicants), which create damage throughout the genome, KR produces spatially limited oxidative DNA damage only in its immediate proximity. This property of KR allows us to engineer oxidative damage specifically at the telomere in a light dose-dependent manner. Using the KR-TEL system to determine the DNA damage response and repair mechanisms at telomeres has several advantages, which make it an ideal system to investigate the mechanism of how telomere integrity is maintained and how this mechanism plays a role in cancer biology.


DNA damage response; DNA strand breaks; KillerRed; Oxidative DNA damage; TRF1; Telomere

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