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Methods. 2017 Feb 1;114:54-59. doi: 10.1016/j.ymeth.2016.07.010. Epub 2016 Aug 1.

In vivo imaging of DNA double-strand break induced telomere mobility during alternative lengthening of telomeres.

Author information

1
Department of Cancer Biology, Abramson Family Cancer Research Institute, Basser Research Center for BRCA, Perelman School of Medicine, University of Pennsylvania, 421 Curie Blvd, Philadelphia, PA 19104-6160, United States. Electronic address: namwcho@mail.med.upenn.edu.
2
Department of Biology, University of Pennsylvania, Philadelphia, PA 19104, United States.
3
Department of Cancer Biology, Abramson Family Cancer Research Institute, Basser Research Center for BRCA, Perelman School of Medicine, University of Pennsylvania, 421 Curie Blvd, Philadelphia, PA 19104-6160, United States; Department of Pathology, Abramson Family Cancer Research Institute, Basser Research Center for BRCA, Perelman School of Medicine, University of Pennsylvania, 421 Curie Blvd, Philadelphia, PA 19104-6160, United States. Electronic address: rogergr@mail.med.upenn.edu.

Abstract

Repair of DNA double-strand breaks (DSBs) by homologous recombination (HR) requires mobilization of chromatin for homology searches that allow interaction of the sequence to be repaired and its template DNA. Here we describe a system to rapidly induce DSBs at telomeres and track their movement, as well as a semi-automated workflow for quantitative analysis. We have successfully used this approach to show that DSBs targeted to telomeres in cells utilizing the alternative lengthening of telomeres (ALT) mechanism increase their diffusion and subsequent long-range directional movement to merge with telomeres on other chromosomes. These methods are simple to implement and are compatible with almost any cell line or in vivo microscopy setup. The magnitude of DSB-induced telomere mobility allows the investigator to easily test for factors regulating telomere mobility during ALT.

KEYWORDS:

ALT; Chromatin mobility; Confocal microscopy; Homologous recombination; Live cell imaging

PMID:
27491801
PMCID:
PMC5378164
DOI:
10.1016/j.ymeth.2016.07.010
[Indexed for MEDLINE]
Free PMC Article

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