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Analysis of De Novo Telomere Addition by Southern Blot

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Genome Instability

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1672))

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

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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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Authors and Affiliations

  1. Institute for Molecular Biology (IMB) gGMBH, Ackermannweg 4, 55128, Mainz, Germany

    Diego Bonetti

  2. Dipartimento di Biotecnologie e Bioscienze, Università di Milano-Bicocca, Milano, Italy

    Diego Bonetti & Maria Pia Longhese

Authors
  1. Diego Bonetti
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  2. Maria Pia Longhese
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Corresponding author

Correspondence to Diego Bonetti .

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Editors and Affiliations

  1. Dipartimento di Bioscienze, Università degli Studi di Milano, Milano, Milano, Italy

    Marco Muzi-Falconi

  2. Donnelly Centre and Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada

    Grant W Brown

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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

  • Print ISBN: 978-1-4939-7305-7

  • Online ISBN: 978-1-4939-7306-4

  • eBook Packages: Springer Protocols

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