Biology subjects

Mazzucco, G.

Publications and source records attributed to Mazzucco, G..

3 recordsLinked to original sources

bioRxiv2025

Inhibition of lagging strand replication by G-rich telomeric DNA and the shelterin subunit POT1

Telomeres preserve stable eukaryotic chromosomes by protecting the natural chromosome ends from DNA repair but pose a persistent challenge to the replication machinery and an endogenous source of replication stress. Different features have been implicated in causing this effect but how the canonical replication process is altered at telomeres in mechanistic terms remains poorly understood. To address this question, we have reconstituted telomere replication with purified human proteins. This system reveals that while G-rich telomeric DNA directly and specifically blocks lagging strand replication in a manner counteracted by BLM helicase, shelterin unexpectedly acts as an additional lagging strand barrier. Biochemical experiments and electron microscopy imaging show that POT1-containing shelterin complexes induce Okazaki fragment skipping by binding the lagging strand template, generating large single-stranded gaps that remain unreplicated. Our study defines how the core components of telomeres interfere with the canonical replication process, identifying several potential sources of replication stress.

molecular biology↗

bioRxiv2021

A method to enrich and purify centromeric DNA from human cells

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, which can be extremely costly. To bypass these issues, we have developed a technique based on selective restriction digestion and size fractionation to enrich for centromeric DNA from human cells. Combining enzymes capable of cutting at high frequency throughout the genome, except within most human centromeres, with size-selection of >20 kb fragments resulted in over 25-fold enrichment in centromeric DNA. Sequencing of the enriched fractions revealed that up to 60% of the enriched material is made of centromeric DNA. This approach has great potential for making sequencing of centromeric DNA more affordable and efficient and for single DNA molecule studies.

molecular biology↗

bioRxiv2020

Telomere damage induces internal loops that generate telomeric circles

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. 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 with the accumulation of extrachromosomal telomeric circles and telomere erosion.

molecular biology↗