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Here we provide a detailed protocol for the enrichment of telomeric repeats from mouse and human cells. The procedure consists of two successive rounds of digestion with frequently cutting restriction enzymes followed by size fractionation. Around 2 mg of genomic DNA is required, and the procedure lasts 5-6 d and yields preparations enriched800-fold in telomeres. The purified material is suitable for single-molecule analysis of telomere structure, visualizing telomere replication and recombination intermediates by electron microscopy or performing molecular combing at telomeric repeats. No special skills are required for the enrichment procedure, while some assistance is needed in harvesting a large number of plates in a timely fashion at the beginning of the procedure. A smaller-scale version of the protocol that involves one round of digestion and purification requires 200 µg of DNA and enriches telomeres ~50-fold in 4 d is also provided. The latter can be combined with specific labeling for single-molecule analysis of replicating DNA or for long-read sequencing analysis of telomeric repeats. The procedure described here can be adapted to the enrichment of other repetitive elements, based on the use of restriction enzymes that do not cut into the repeat of interest.

Extrachromosomal telomeric circles are commonly invoked as important players in telomere maintenance, but their origin has remained elusive. Using electron microscopy analysis on purified telomeres we show that, apart from known structures, telomeric repeats accumulate internal loops (i-loops) that occur in the proximity of nicks and single-stranded DNA gaps. I-loops are induced by single-stranded damage at normal telomeres and represent the majority of telomeric structures detected in ALT (Alternative Lengthening of Telomeres) tumor cells. Our data indicate that i-loops form as a consequence of the exposure of single-stranded DNA at telomeric repeats. Finally, we show that these damage-induced i-loops can be excised to generate extrachromosomal telomeric circles resulting in loss of telomeric repeats. Our results identify damage-induced i-loops as a new intermediate in telomere metabolism and reveal a simple mechanism that links telomere damage to the accumulation of extrachromosomal telomeric circles and to telomere erosion., Extrachromosomal circular DNA made of telomeric repeats have been found to have an effect on telomere maintenance. By combining electron microscopy with a telomere purification procedure the authors identify damage-induced i-loops as a key intermediate in telomere circle formation.