Abstract
Base excision repair (BER) is a major pathway for the removal of endogenous and exogenous DNA damage. This repair mechanism is initiated by DNA glycosylases that excise the altered base, and continues through alternative routes that culminate in DNA resynthesis and ligation. In contrast to the information available for microbes and animals, our knowledge about this important DNA repair pathway in plants is very limited, partially due to a lack of biochemical approaches. Here we describe an in vitro assay to monitor BER in cell-free extracts from the model plant Arabidopsis thaliana. The assay uses labeled DNA substrates containing a single damaged base within a restriction site, and allows detection of fully repaired molecules as well as DNA repair intermediates. The method is easily applied to measure the repair activity of purified proteins and can be successfully used in combination with the extensive array of biological resources available for Arabidopsis.
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Acknowledgements
We thank members of our laboratory for helpful discussion and advice. This work was supported by the Spanish Ministry of Science and Innovation and the European Regional Development Fund (grant BFU2010-18838), and by the Junta de Andalucía (grant P07-CVI-02770).
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Córdoba-Cañero, D., Roldán-Arjona, T., Ariza, R.R. (2012). Using Arabidopsis Cell Extracts to Monitor Repair of DNA Base Damage In Vitro. In: Bjergbæk, L. (eds) DNA Repair Protocols. Methods in Molecular Biology, vol 920. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-998-3_18
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DOI: https://doi.org/10.1007/978-1-61779-998-3_18
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Publisher Name: Humana Press, Totowa, NJ
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