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

- mCherry-ER-DD-TRF1-FokI is used to visualize DSB-induced telomere mobility.
- DSB generation and tracking are performed using a single protein.
- Transfection and imaging can be accomplished in as little as 48 h.
- Telomere mobility can be analyzed quantitatively using a semi-automated workflow.

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.