Telomere damage induces internal loops that generate telomeric circles

Abstract

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 proximity of nicks and single-stranded DNA gaps. Iloops 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 singlestranded 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 with the accumulation of extrachromosomal telomeric circles and telomere erosion.

A method to enrich and purify centromeric DNA from human cells

“…This is even more important in techniques in which the usage of fluorescent probes is not feasible as EM or atomic force microscopy. For example, following in vivo psoralen crosslinking, the enriched centromeric DNA can be further processed for EM to study the topological structures and replication intermediates at centromeres, as done for telomeres (Mazzucco et al, 2020;Griffith et al, 1999). This has the potential to shed light on the architecture and replication intermediates that are present at centromeric regions and to understand how centromeric DNA binding proteins might modulate their topology and structure.…”

“…Genomic DNA was extracted from 300-400 million cells as described in (Mazzucco et al, 2020) Briefly:…”

“…It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted September 24, 2021. ; https://doi.org/10.1101/2021.09.24.461328 doi: bioRxiv preprint hybridized 1 hour at 65° in Church mix (500 mM NaPi pH 7.2, 1 mM EDTA pH 8.0, 7% SDS, 1% BSA). Hybridization occurred overnight in Church mix with a telomeric TTAGGG probe (Mazzucco et al, 2020) or centromeric probe (produced as described below). After three washes in Church wash buffer (40 mM NaPi pH 7.2, 1 mM EDTA pH 8.0, 1% SDS), radioactive signal was impressed on a FUJIFILM Storage Phosphor screen for 5 hours and acquired with Typhon Trio (GE healthcare).…”

“…More recently, a two-step procedure has been developed for the study of telomere structure by electron microscopy (EM) (Mazzucco et al, 2020). While a similar restriction-based approach was developed in the pre-genomic era to isolate mouse (peri)centromeres (Lica & Hamkalo, 1983), an analogous technique for the study of human centromeres is currently missing.…”

Charakterystyka kolistych DNA u Eukarya

“…Podczas podziału większość kolistych rDNA jest zatrzymywana w komórkach matczynych. Natomiast w komórkach potomnych populację rDNA circle tworzą głównie cząsteczki uformowane później [39,46]. Zauważono, że w drożdżach pozbawionych helikazy Sgs1 chroniącej DNA przed uszkodzeniami (pofragmentowaniem), koliste rDNA ulegają szybszemu i większemu nagromadzeniu, niż w organizmach o fenotypie dzikim [39].…”