Abstract
Aberrant transcription induced by DNA damage may confer risk for the development of cancer and other human diseases. Traditional methods for measuring lesion-induced transcriptional alterations often involve extensive colony screening and DNA sequencing procedures. Here we describe a protocol for the quantitative assessment of the effects of DNA lesions on the efficiency and fidelity of transcription in vitro and in mammalian cells. The method is also amenable to investigating the influence of specific DNA repair proteins on the biological response toward DNA damage during transcription by manipulating their gene expression. Specifically, we present detailed, step-by-step procedures, including DNA template preparation, in vitro and in vivo transcription, RNA purification, reverse-transcription PCR (RT-PCR) and restriction digestion of RT-PCR products. Analyses of restriction fragments of interest are performed by liquid chromatography–tandem mass spectrometry (LC-MS/MS) and polyacrylamide gel electrophoresis (PAGE). The entire procedure described in this protocol can be completed in 15–20 d.
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Acknowledgements
We acknowledge T.R. O'Connor (The City of Hope) and K.A. Cimprich (Stanford University) for kindly providing the initial pTGFP-Hha10 vector and human fibroblast cells (XP12RO and GM15876A), respectively. This work was supported by US National Institutes of Health (R01DK082779, R01ES019873, R01 ES025121 and R01CA101864 to Y.W.).
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C.Y. designed and performed the research and wrote the manuscript. Y.W. designed and supervised the research and edited the manuscript.
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You, C., Wang, Y. Quantitative measurement of transcriptional inhibition and mutagenesis induced by site-specifically incorporated DNA lesions in vitro and in vivo. Nat Protoc 10, 1389–1406 (2015). https://doi.org/10.1038/nprot.2015.094
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DOI: https://doi.org/10.1038/nprot.2015.094
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