In vivo measurements of interindividual differences in DNA glycosylases and APE1 activities
Autor: | Jennifer J. Jordan, Patrizia Mazzucato, Leona D. Samson, Zachary D. Nagel, Le P. Ngo, Isaac A. Chaim |
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Rok vydání: | 2017 |
Předmět: |
0301 basic medicine
DNA Repair DNA damage DNA repair T-Lymphocytes Primary Cell Culture Biology Models Biological Cell Line DNA Glycosylases 03 medical and health sciences 0302 clinical medicine DNA-(Apurinic or Apyrimidinic Site) Lyase Humans AP site Precision Medicine RNA Small Interfering Multidisciplinary Base excision repair DNA Hydrogen Peroxide Flow Cytometry Methyl Methanesulfonate DNA-(apurinic or apyrimidinic site) lyase Healthy Volunteers 030104 developmental biology Biochemistry Biological Variation Population PNAS Plus DNA glycosylase Mutagenesis 030220 oncology & carcinogenesis Uracil-DNA glycosylase Gene Knockdown Techniques Fluorouracil Single-Cell Analysis Nucleotide excision repair DNA Damage Mutagens |
Zdroj: | Proceedings of the National Academy of Sciences of the United States of America. 114(48) |
ISSN: | 1091-6490 |
Popis: | The integrity of our DNA is challenged with at least 100,000 lesions per cell on a daily basis. Failure to repair DNA damage efficiently can lead to cancer, immunodeficiency, and neurodegenerative disease. Base excision repair (BER) recognizes and repairs minimally helix-distorting DNA base lesions induced by both endogenous and exogenous DNA damaging agents. Levels of BER-initiating DNA glycosylases can vary between individuals, suggesting that quantitating and understanding interindividual differences in DNA repair capacity (DRC) may enable us to predict and prevent disease in a personalized manner. However, population studies of BER capacity have been limited because most methods used to measure BER activity are cumbersome, time consuming and, for the most part, only allow for the analysis of one DNA glycosylase at a time. We have developed a fluorescence-based multiplex flow-cytometric host cell reactivation assay wherein the activity of several enzymes [four BER-initiating DNA glycosylases and the downstream processing apurinic/apyrimidinic endonuclease 1 (APE1)] can be tested simultaneously, at single-cell resolution, in vivo. Taking advantage of the transcriptional properties of several DNA lesions, we have engineered specific fluorescent reporter plasmids for quantitative measurements of 8-oxoguanine DNA glycosylase, alkyl-adenine DNA glycosylase, MutY DNA glycosylase, uracil DNA glycosylase, and APE1 activity. We have used these reporters to measure differences in BER capacity across a panel of cell lines collected from healthy individuals, and to generate mathematical models that predict cellular sensitivity to methylmethane sulfonate, H2O2, and 5-FU from DRC. Moreover, we demonstrate the suitability of these reporters to measure differences in DRC in multiple pathways using primary lymphocytes from two individuals. |
Databáze: | OpenAIRE |
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