Abstract
Mapping the precise position of endonucleolytic cleavage sites is a fundamental experimental technique used to describe the function of a homing endonuclease. However, these proteins are often recalcitrant to cloning and over-expression in biological systems because of toxicity induced by spurious DNA cleavage events. In this chapter we outline the steps to successfully express a homing endonuclease in vitro and use this product in nucleotide-resolution cleavage assays.
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Acknowledgements
We would like to thank Caren J. Stark, Matthew Stanger, Dorie Smith, and Carol Lyn Piazza for critical reading of the manuscript. Research in the Belfort Lab is supported by NIH grants GM39422 and GM44844.
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Bonocora, R.P., Belfort, M. (2014). Mapping Homing Endonuclease Cleavage Sites Using In Vitro Generated Protein. In: Edgell, D. (eds) Homing Endonucleases. Methods in Molecular Biology, vol 1123. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-968-0_4
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DOI: https://doi.org/10.1007/978-1-62703-968-0_4
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