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In vivo measurements of interindividual differences in DNA glycosylases and APE1 activities
- Source :
- Proceedings of the National Academy of Sciences of the United States of America. 114(48)
- Publication Year :
- 2017
-
Abstract
- 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.
- Subjects :
- 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
Subjects
Details
- ISSN :
- 10916490
- Volume :
- 114
- Issue :
- 48
- Database :
- OpenAIRE
- Journal :
- Proceedings of the National Academy of Sciences of the United States of America
- Accession number :
- edsair.doi.dedup.....907e26ce8c51d12199d0e20d8372df43