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In vivo measurements of interindividual differences in DNA glycosylases and APE1 activities

  1. Leona D. Samsona,b,c,d,1
  1. aDepartment of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139;
  2. bCenter for Environmental Health Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139;
  3. cDepartment of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139;
  4. dThe David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139
  1. Edited by Paul Modrich, Howard Hughes Medical Institute and Duke University Medical Center, Durham, NC, and approved October 20, 2017 (received for review July 6, 2017)

Significance

The DNA in each cell is damaged thousands of times daily. Consequently, a battery of DNA repair pathways exist that allow repair of this damage. Failure to repair can lead to devastating diseases, including cancer and neurodegeneration. Each individual’s DNA repair capacity (DRC) is inherently different. Being able to measure an individual’s DRC could contribute to a personalized approach to prevent and treat disease. Here we present powerful tools for measuring in vivo base excision repair capacity for five distinct DNA lesions. We use these methods to predict the cellular responses to a variety of DNA damaging agents, and to monitor differences in DRC in primary human lymphocytes. Additionally, we unveil previously unknown transcriptional mutagenesis induced by DNA lesions.

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.

Footnotes

  • ?1To whom correspondence should be addressed. Email: lsamson{at}mit.edu.
  • Author contributions: I.A.C. and L.D.S. designed research; I.A.C., Z.D.N., J.J.J., P.M., and L.P.N. performed research; I.A.C. contributed new reagents/analytic tools; I.A.C. and J.J.J. analyzed data; and I.A.C., Z.D.N., and L.D.S. wrote the paper.

  • The authors declare no conflict of interest.

  • This article is a PNAS Direct Submission.

  • This article contains supporting information online at www.danielhellerman.com/lookup/suppl/doi:10.1073/pnas.1712032114/-/DCSupplemental.

Published under the PNAS license.

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