Abstract
Telomere length is maintained in most eukaryotes by the action of a specialized enzyme, the telomerase. However, the complexity of mechanisms regulating telomeric DNA length as well as the heterogeneity in length of each telomere in a population of cells has made it very difficult to understand how telomerase is regulated in vivo. Here, we describe a method developed in Saccharomyces cerevisiae to monitor the addition of telomeric sequences to a single newly generated telomere in vivo. The primary strain consists of a HO endonuclease cleavage site that is placed directly adjacent to an 81-base-pair stretch of telomeric DNA inserted into the ADH4 locus of chromosome VII. Upon cleavage by HO, the de novo DNA end is rapidly healed by the telomerase enzyme and the analysis of this process allows to gain a mechanistic understanding of how telomerase action is regulated in the cell.
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References
Bonetti D, Martina M, Falcettoni M, Longhese MP (2014) Telomere-end processing: mechanisms and regulation. Chromosoma 123:57–66
Wellinger RJ, Zakian VA (2012) Everything you ever wanted to know about Saccharomyces cerevisiae telomeres: beginning to end. Genetics 191:1073–1105
Marcand S, Brevet V, Gilson E (1999) Progressive cis-inhibition of telomerase upon telomere elongation. EMBO J 18:3509–3519
Teixeira MT, Arneric M, Sperisen P, Lingner J (2004) Telomere length homeostasis is achieved via a switch between telomerase- extendible and -nonextendible states. Cell 117:323–335
Bianchi A, Shore D (2007) Increased association of telomerase with short telomeres in yeast. Genes Dev 21:1726–1730
Diede SJ, Gottschling DE (1999) Telomerase-mediated telomere addition in vivo requires DNA primase and DNA polymerase α and δ. Cell 99:723–733
Diede SJ, Gottschling DE (2001) Exonuclease activity is required for sequence addition and Cdc13p loading at a de novo telomere. Curr Biol 11:1336–1340
Kramer KM, Haber JE (1993) New telomeres in yeast are initiated with a highly selected subset of TG1-3 repeats. Genes Dev 7:2345–2356
Acknowledgments
We thank G. Lucchini for critical reading of the manuscript. D.B. has been supported by a fellowship from CancerTelSys (grant 01ZX1302) in the E:med program of the German Federal Ministry of Education and Research (BMBF). Research in Longhese’s lab is supported by Associazione Italiana per la Ricerca sul Cancro (AIRC) (grant number 15210) and Cofinanziamento 2015 MIUR/Università di Milano-Bicocca.
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Bonetti, D., Longhese, M.P. (2018). Analysis of De Novo Telomere Addition by Southern Blot. In: Muzi-Falconi, M., Brown, G. (eds) Genome Instability. Methods in Molecular Biology, vol 1672. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7306-4_25
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DOI: https://doi.org/10.1007/978-1-4939-7306-4_25
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Publisher Name: Humana Press, New York, NY
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Online ISBN: 978-1-4939-7306-4
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