Purification of mammalian telomeric DNA for single-molecule analysis

Mazzucco, Giulia; Huda, Armela; Galli, Maria Grazia; Zanella, Elia; Doksani, Ylli

doi:10.1038/s41596-022-00684-9

Cited by 6 publications

(11 citation statements)

References 33 publications

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“…Telomeres from this cell line were enriched through two successive rounds of restriction digestion with frequent cutters, followed by size fractionation ( Fig. 4A ) ( 31 ). For EM analysis, Y-shaped molecules (replication forks) and X-shaped intermediates with two equal arms (compatible with fork reversal) were quantified in parallel in the nonenriched (bulk) DNA and after telomere enrichment (telomeric) ( Fig.…”

Section: Resultsmentioning

confidence: 99%

The telomerase reverse transcriptase elongates reversed replication forks at telomeric repeats

et al. 2023

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The telomerase reverse transcriptase elongates telomeres to prevent replicative senescence. This process requires exposure of the 3′-end, which is thought to occur when two sister telomeres are generated at replication completion. Using two-dimensional agarose gel electrophoresis (2D-gels) and electron microscopy, we found that telomeric repeats are hotspots for replication fork reversal. Fork reversal generates 3′ telomeric ends before replication completion. To verify whether these ends are elongated by telomerase, we probed de novo telomeric synthesis in situ and at replication intermediates by reconstituting mutant telomerase that adds a variant telomere sequence. We found variant telomeric repeats overlapping with telomeric reversed forks in 2D-gels, but not with normal forks, nontelomeric reversed forks, or telomeric reversed forks with a C-rich 3′-end. Our results define reversed telomeric forks as a substrate of telomerase during replication.

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Section: Resultsmentioning

confidence: 99%

The telomerase reverse transcriptase elongates reversed replication forks at telomeric repeats

et al. 2023

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“…To obtain a centromere-enriched sample to be analyzed with multiple techniques, 2.5 to 3 mg of genomic DNA was extracted from 300–400 million cells, as previously described [ 18 , 19 ]. When less enriched DNA is needed, (e.g.…”

Section: Description Of the Methodsmentioning

confidence: 99%

“…This rationale is applied for the purification of telomeric repeats, which lack canonical restriction sites [ 15 – 17 ]. More recently, a two-step procedure has been developed for the study of telomere structure by electron microscopy (EM) [ 18 , 19 ]. While a similar restriction-based approach was developed in the pre-genomic era to isolate mouse (peri)centromeres [ 20 ], an analogous widely established technique for the study of human centromeres is currently missing.…”

Section: Introductionmentioning

confidence: 99%

Enrichment of centromeric DNA from human cells

et al. 2022

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Centromeres are key elements for chromosome segregation. Canonical centromeres are built over long-stretches of tandem repetitive arrays. Despite being quite abundant compared to other loci, centromere sequences overall still represent only 2 to 5% of the human genome, therefore studying their genetic and epigenetic features is a major challenge. Furthermore, sequencing of centromeric regions requires high coverage to fully analyze length and sequence variations, and this can be extremely costly. To bypass these issues, we have developed a technique, named CenRICH, to enrich for centromeric DNA from human cells based on selective restriction digestion and size fractionation. Combining restriction enzymes cutting at high frequency throughout the genome, except within most human centromeres, with size-selection of fragments >20 kb, resulted in over 25-fold enrichment in centromeric DNA. High-throughput sequencing revealed that up to 60% of the DNA in the enriched samples is made of centromeric repeats. We show that this method can be used in combination with long-read sequencing to investigate the DNA methylation status of certain centromeres and, with a specific enzyme combination, also of their surrounding regions (mainly HSATII). Finally, we show that CenRICH facilitates single-molecule analysis of replicating centromeric fibers by DNA combing. This approach has great potential for making sequencing of centromeric DNA more affordable and efficient and for single DNA molecule studies.

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“…The incubation and irradiation steps were repeated three more times (4 cycles total). Genomic DNA (gDNA) was extracted with phenol-chloroform as described in 66 . 50 µg of gDNA were digested with KpnI and passed through a QIAGEN Genomic-tip 20/G column (QIAGEN) to enrich for replication intermediates, as described by Zellweger and Lopes 67 .…”

Section: Methodsmentioning

confidence: 99%

“…For EM analysis, hTERT RPE-1 cells were pulsed for 24 hours with reversine or vehicle control (DMSO) and then harvested. Immediately after, cells were psoralen-crosslinked in vivo to stabilize replication intermediates as described in 66 . The cell suspension was first incubated with 30 µg/ml 4, 5′, 8-trimethylpsoralen (2mg/ml, Sigma) for 5 min in the dark and then exposed to 365 nm UV light for 8 min in a UV Stratalinker 1800, (Stratagene), with 365 nm UV bulbs (model UVL-56, UVP) at 2-3 cm from the light source.…”

Section: Ultra-structural Analysis Of Replication Intermediatesmentioning

confidence: 99%

Short-term molecular consequences of chromosome mis-segregation for genome stability

Feudis

Garribba

Martis

et al. 2022

Preprint

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Genome instability is a hallmark of cancer. The most common form of genome instability is chromosomal instability (CIN), a condition in which cells mis-segregate their chromosomes during cell division. CIN leads to aneuploidy, a state of karyotype imbalance, often found in tumors. Although the causal relationship between CIN and aneuploidy is well established, evidence is limited for a direct involvement of aneuploidy in promoting CIN. Here, we show that aneuploid cells experience DNA replication stress in their first S-phase and precipitate in a state of continuous CIN, eventually accumulating complex karyotypes. Mechanistically, we find that aneuploid cells fire dormant replication origins through a Dbf4-dependent kinase (DDK)-driven mechanism and complete replication of genomic loci through mitotic DNA synthesis (MiDAS). By following the fate of aneuploid cells, we also show that, when they divide, DNA damage can be distributed asymmetrically between daughter cells, and this may partially explain why some aneuploid cells are able to continue proliferating and others stop dividing. We further found that cycling aneuploid cells display lower karyotype complexity compared to arrested ones and increased expression of gene signatures associated to DNA repair. Interestingly, by stratifying aneuploid human cancer cells by their doubling times, we found the same DNA repair signatures to be upregulated in highly-proliferative cancer cells, which might enable them to keep proliferating despite the disadvantage conferred by aneuploidy-induced genome instability. In summary, our study reveals the origins of genome instability following induction of aneuploidy and indicates the aneuploid state of cancer cells as a point mutation-independent source of genome instability, providing an explanation for the high occurrence of aneuploidy in tumors.

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Purification of mammalian telomeric DNA for single-molecule analysis

Cited by 6 publications

References 33 publications

The telomerase reverse transcriptase elongates reversed replication forks at telomeric repeats

The telomerase reverse transcriptase elongates reversed replication forks at telomeric repeats

Enrichment of centromeric DNA from human cells

Short-term molecular consequences of chromosome mis-segregation for genome stability

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