Detection of Telomeric DNA:RNA Hybrids Using TeloDRIP-qPCR

Abstract

Because of their intrinsic characteristics, telomeres are genomic loci that pose significant problems during the replication of the genome. In particular, it has been observed that telomeres that are maintained in cancer cells by the alternative mechanism of the lengthening of telomeres (ALT) harbor higher levels of replicative stress compared with telomerase-positive cancer cells. R-loops are three-stranded structures formed by a DNA:RNA hybrid and a displaced ssDNA. Emerging evidence suggests that controlling the levels of R-loops at ALT telomeres is critical for telomere maintenance. In fact, on the one hand, they favor telomere recombination, but on the other, they are a source of detrimental replicative stress. DRIP (DNA:RNA immunoprecipitation) is the main technique used for the detection of R-loops, and it is based on the use of the S9.6 antibody, which recognizes preferentially DNA:RNA hybrids in a sequence-independent manner. The detection of DNA:RNA hybrids in repetitive sequences such as telomeres requires some additional precautions as a result of their repetitive nature. Here, we share an optimized protocol for the detection of telomeric DNA:RNA hybrids, and we demonstrate its application in an ALT and in a telomerase-positive cell line. We demonstrate that ALT telomeres bear higher levels of DNA:RNA hybrids, and we propose this method as a reliable way to detect them in telomeres.

Transcription stress at telomeres leads to cytosolic DNA release and paracrine senescence

“…The complexes were eluted, and the crosslinking was heat reversed. Purified DNA fragments were analysed by qPCR using sets of primers targeting telomeric repeats as previously described 67 . For yeast Rap1 ChIP, cells were grown in exponential phase and cross-linked with 1.2% formaldehyde for 10 min at RT and quenched with 115 mM glycine for 5 min.…”

“…The beads–antibody–DNA complexes were then disrupted by incubation with elution buffer (50 mM Tris-HCl pH8.0, 10 mM EDTA pH8.0, 1% SDS, 0.5 mg/ml proteinase K) overnight at 37 o C, and 1 h at 52 o C. The immunoprecipitated DNA was collected and purified by using MinElute PCR Purification kit (28004, QIAGEN). Purified DNA fragments were analysed by qPCR using sets of primers targeting telomeric repeats as previously described 67 . All the steps of OxiDIP-protocol were carried out in low-light conditions.…”

“…Telo-DRIP experiments were performed as previously described 67 . Briefly, MEFs were harvested by scraping and lysed in 1 mL Tris-EDTA, 0.5% sodium dodecyl sulfate (SDS), and 200 µg/mL proteinase K (25530049, Invitrogen) in presence of RNAseOUT (10777019, Invitrogen) overnight at 37 °C at 350 rpm.…”

“…The supernatants were recovered, and the DNA was precipitated and resuspended in Tris-EDTA. Purified RNA-DNA fragments were analysed by qPCR using sets of primers targeting telomeric repeats, as previously described 67 . ChIP, Oxi-DIP and Telo-DRIP signals in the figures are shown as % of input after normalizing with IgG and IgM negative corresponding controls.…”