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J Biol Chem. 2015 Nov 27;290(48):28812-21. doi: 10.1074/jbc.M115.690354. Epub 2015 Oct 5.

An Integrated Approach for Analysis of the DNA Damage Response in Mammalian Cells: NUCLEOTIDE EXCISION REPAIR, DNA DAMAGE CHECKPOINT, AND APOPTOSIS.

Author information

1
From the Center for Bioanalysis, Department of Metrology for Quality of Life, Korea Research Institute of Standards and Science, Daejeon 305-340, South Korea, the Department of Bio-Analytical Science, University of Science & Technology, Daejeon 305-350, South Korea, and junchoi@kriss.re.kr.
2
From the Center for Bioanalysis, Department of Metrology for Quality of Life, Korea Research Institute of Standards and Science, Daejeon 305-340, South Korea.
3
From the Center for Bioanalysis, Department of Metrology for Quality of Life, Korea Research Institute of Standards and Science, Daejeon 305-340, South Korea, the Department of Bio-Analytical Science, University of Science & Technology, Daejeon 305-350, South Korea, and.
4
the Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599-7260.
5
the Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599-7260 aziz_sancar@med.unc.edu.

Abstract

DNA damage by UV and UV-mimetic agents elicits a set of inter-related responses in mammalian cells, including DNA repair, DNA damage checkpoints, and apoptosis. Conventionally, these responses are analyzed separately using different methodologies. Here we describe a unified approach that is capable of quantifying all three responses in parallel using lysates from the same population of cells. We show that a highly sensitive in vivo excision repair assay is capable of detecting nucleotide excision repair of a wide spectrum of DNA lesions (UV damage, chemical carcinogens, and chemotherapeutic drugs) within minutes of damage induction. This method therefore allows for a real-time measure of nucleotide excision repair activity that can be monitored in conjunction with other components of the DNA damage response, including DNA damage checkpoint and apoptotic signaling. This approach therefore provides a convenient and reliable platform for simultaneously examining multiple aspects of the DNA damage response in a single population of cells that can be applied for a diverse array of carcinogenic and chemotherapeutic agents.

KEYWORDS:

DNA damage; DNA damage checkpoint; DNA damage response; DNA repair; apoptosis; carcinogenesis; cell signaling; checkpoint control; chemotherapy; nucleotide excision repair

PMID:
26438822
PMCID:
PMC4661397
DOI:
10.1074/jbc.M115.690354
[Indexed for MEDLINE]
Free PMC Article

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