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.
De novo telomere HO endonuclease S. cerevisiaeSouthern blot Telomerase
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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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