Visualization and quantitative analysis of extrachromosomal telomere-repeat DNA in individual human cells by Halo-FISH

Abstract

Current methods for characterizing extrachromosomal nuclear DNA in mammalian cells do not permit single-cell analysis, are often semi-quantitative and frequently biased toward the detection of circular species. To overcome these limitations, we developed Halo-FISH to visualize and quantitatively analyze extrachromosomal DNA in single cells. We demonstrate Halo-FISH by using it to analyze extrachromosomal telomere-repeat (ECTR) in human cells that use the Alternative Lengthening of Telomeres (ALT) pathway(s) to maintain telomere lengths. We find that GM847 and VA13 ALT cells average ∼80 detectable G/C-strand ECTR DNA molecules/nucleus, while U2OS ALT cells average ∼18 molecules/nucleus. In comparison, human primary and telomerase-positive cells contain <5 ECTR DNA molecules/nucleus. ECTR DNA in ALT cells exhibit striking cell-to-cell variations in number (<20 to >300), range widely in length (<1 to >200 kb) and are composed of primarily G- or C-strand telomere-repeat DNA. Halo-FISH enables, for the first time, the simultaneous analysis of ECTR DNA and chromosomal telomeres in a single cell. We find that ECTR DNA comprises ∼15% of telomere-repeat DNA in GM847 and VA13 cells, but <4% in U2OS cells. In addition to its use in ALT cell analysis, Halo-FISH can facilitate the study of a wide variety of extrachromosomal DNA in mammalian cells.

Clustered telomeres in phase-separated nuclear condensates engage mitotic DNA synthesis through BLM and RAD52

“…Indeed, overexpression of BLM WT in the absence of telomere clustering scaffolds did not induce any changes in G-rich single-stranded telomere DNA (Supplemental Fig S9A-C). We interpret these data to support the idea that single-stranded telomere DNA generation processes are involved in the induction of the ALT phenotypes (Nabetani and The helicase activity of BLM may be required for the association of ECTRs in APBs with chromosomal telomeres in response to DNA replication stresses (Komosa et al 2015;Root et al 2016). Moreover, overexpression of telomere clustering scaffolds induces persistent telomere clustering, leading to G2 and M arrest (Supplemental Fig.…”

Telomeres are partly shielded from ultraviolet-induced damage and proficient for nucleotide excision repair of photoproducts

“…U2OS cells use the alternative lengthening of telomeres (ALT) pathway, and ALT cells contain extrachromosomal telomere-repeat (ECTR) DNA. However, ECTR comprises <4% of the telomeric repeat DNA in U2OS cells 38 . Of the ECTR species, G-circles consisting of single-stranded TTAGGG repeats could potentially anneal with the telomere capture oligonucleotide.…”

Alternative Lengthening of Telomeres Mediated by Mitotic DNA Synthesis Engages Break-Induced Replication Processes

“…Moreover, these are also increased in response to double-strand breaks or stalled replication forks at telomeres (11,(40)(41)(42)(43)(44)(45)(46)(47)(48). It has also been previously reported that large telomeric foci contain extrachromosomal telomeric DNAs (49). These extrachromosomal telomeric DNAs can form large telomeric foci with chromosomal DNAs in response to DNA replication stress in a BLM helicase-dependent manner (50).…”