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Analysis of De Novo Telomere Addition by Southern Blot

  • Diego BonettiEmail author
  • Maria Pia Longhese
Protocol
Part of the Methods in Molecular Biology book series (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.

Key words

De novo telomere HO endonuclease S. cerevisiae Southern blot Telomerase 

Notes

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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Copyright information

© Springer Science+Business Media LLC 2018

Authors and Affiliations

  1. 1.Institute for Molecular Biology (IMB) gGMBHMainzGermany
  2. 2.Dipartimento di Biotecnologie e BioscienzeUniversità di Milano-BicoccaMilanoItaly

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