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Telomere Terminal G/C Strand Synthesis: Measuring Telomerase Action and C-Rich Fill-In

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Telomeres and Telomerase

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1587))

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Abstract

Telomerase is present in most human cancers, and proliferative stem cells including germline cells. Telomerase plays an essential role in tumorigenesis by maintaining/elongating telomeric DNA, and thus preventing the telomere shortening that results in replicative senescence. Understanding telomerase action in vivo has important implication for both cancer and aging, but there are not robust methods for monitoring telomerase action. By combining a series of cell biological and biochemical approaches, and taking advantage of the enzyme DSN that specifically cuts double-stranded DNA and releases the telomeric overhangs, we have developed a method to monitor telomerase action during one cell cycle. Here, we describe this method using Hela carcinoma cells as an example.

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References

  1. Zhao Y, Hoshiyama H, Shay JW, Wright WE (2008) Quantitative telomeric overhang determination using a double-strand specific nuclease. Nucleic Acids Res 36:e14

    Article  PubMed  Google Scholar 

  2. Wright WE, Tesmer VM, Huffman KE, Levene SD, Shay JW (1997) Normal human chromosomes have long G-rich telomeric overhangs at one end. Genes Dev 11:2801–2809

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Smogorzewska A, de Lange T (2004) Regulation of telomerase by telomeric proteins. Annu Rev Biochem 73:177–208

    Article  CAS  PubMed  Google Scholar 

  4. Kim NW, Piatyszek MA, Prowse KR, Harley CB, West MD, Ho PL, Coviello GM, Wright WE, Weinrich SL, Shay JW (1994) Specific association of human telomerase activity with immortal cells and cancer. Science 266:2011–2015

    Article  CAS  PubMed  Google Scholar 

  5. Greider CW, Blackburn EH (1985) Identification of a specific telomere terminal transferase activity in Tetrahymena extracts. Cell 43:405–413

    Article  CAS  PubMed  Google Scholar 

  6. Harley CB, Futcher AB, Greider CW (1990) Telomeres shorten during ageing of human fibroblasts. Nature 345:458–460

    Article  CAS  PubMed  Google Scholar 

  7. Meyne J, Moyzis RK (1994) In situ hybridization using synthetic oligomers as probes for centromere and telomere repeats. Methods Mol Biol 33:63–74

    CAS  PubMed  Google Scholar 

  8. Baird DM, Rowson J, Wynford-Thomas D, Kipling D (2003) Extensive allelic variation and ultrashort telomeres in senescent human cells. Nat Genet 33:203–207

    Article  CAS  PubMed  Google Scholar 

  9. Wright WE, Tesmer VM, Liao ML, Shay JW (1999) Normal human telomeres are not late replicating. Exp Cell Res 251:492–499

    Article  CAS  PubMed  Google Scholar 

  10. Zhao Y, Sfeir AJ, Zou Y, Buseman CM, Chow TT, Shay JW, Wright WE (2009) Telomere extension occurs at most chromosome ends and is uncoupled from fill-in in human cancer cells. Cell 138:463–475

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Chai W, Du Q, Shay JW, Wright WE (2006) Human telomeres have different overhang sizes at leading versus lagging strands. Mol Cell 21:427–435

    Article  PubMed  Google Scholar 

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Correspondence to Woodring E. Wright .

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Zhao, Y., Shay, J.W., Wright, W.E. (2017). Telomere Terminal G/C Strand Synthesis: Measuring Telomerase Action and C-Rich Fill-In. In: Songyang, Z. (eds) Telomeres and Telomerase. Methods in Molecular Biology, vol 1587. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6892-3_7

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  • DOI: https://doi.org/10.1007/978-1-4939-6892-3_7

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6891-6

  • Online ISBN: 978-1-4939-6892-3

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