The Response to DNA Damage at Telomeric Repeats and Its Consequences for Telomere Function

Telomeric repeats, coated by the shelterin complex, prevent inappropriate activation of the DNA damage response at the ends of linear chromosomes. Shelterin has evolved distinct solutions to protect telomeres from different aspects of the DNA damage response. These solutions include formation of t-loops, which can sequester the chromosome terminus from DNA-end sensors and inhibition of key steps in the DNA damage response. While blocking the DNA damage response at chromosome ends, telomeres make wide use of many of its players to deal with exogenous damage and replication stress. This review focuses on the interplay between the end-protection functions and the response to DNA damage occurring inside the telomeric repeats, as well as on the consequences that telomere damage has on telomere structure and function

The role of double-strand break repair pathways at functional and dysfunctional telomeres. Cold Spring Harb Perspect Biol 6(12):a016576.

Telomeres have evolved to protect the ends of linear chromosomes from the myriad of threats posed by the cellular DNA damage signaling and repair pathways. Mammalian telomeres have to block nonhomologous end joining (NHEJ), thus preventing chromosome fusions; they need to control homologous recombination (HR), which could change telomere lengths; they have to avoid activating the ATM (ataxia telangiectasia mutated) and ATR (ATM-and RAD3-related) kinase pathways, which could induce cell cycle arrest; and they have to protect chromosome ends from hyperresection. Recent studies of telomeres have provided insights into the mechanisms of NHEJ and HR, how these double-strand break (DSB) repair pathways can be thwarted, and how telomeres have co-opted DNA repair factors to help in the protection of chromosome ends. These aspects of telomere biology are reviewed here with particular emphasis on recombination, the main focus of this collection

Telomere-Internal Double-Strand Breaks Are Repaired by Homologous Recombination and PARP1/Lig3-Dependent End-Joining

Shelterin protects chromosome ends from the DNA damage response. Although the mechanism of telomere protection has been studied extensively, the fate of double-strand breaks (DSBs) inside telomeres is not known. Here, we report that telomere-internal FokI-induced DSBs activate ATM kinase-dependent signaling in S-phase but are well tolerated and repaired efficiently. Homologous recombination contributes to repair, leading to increased telomere length heterogeneity typical of the alternative lengthening of telomeres (ALT) pathway. Furthermore, cells accumulate extra chromosomal telomeric signals (ECTS), a second hallmark of ALT. Telomere-internal DSBs are also repaired by a PARP1- and Ligase3-dependent reaction, suggesting alternative non-homologous end-joining (alt-NHEJ), which relies on microhomology at DSBs. However, as resected telomere-internal DSBs have perfect homology, their PARP1/Lig3-dependent end-joining may be more akin to single strand break repair. We conclude that shelterin does not repress ATM kinase signaling or DSB repair at telomere-internal sites, thereby allowing DNA repair to maintain telomere integrity

Replicon dynamics, dormant origin firing, and terminal fork integrity after double-strand break formation

In response to replication stress, the Mec1/ATR and SUMO pathways control stalled- and damaged-fork stability. We investigated the S phase response at forks encountering a broken template (termed the terminal fork). We show that double-strand break (DSB) formation can locally trigger dormant origin firing. Irreversible fork resolution at the break does not impede progression of the other fork in the same replicon (termed the sister fork). The Mre11-Tel1/ATM response acts at terminal forks, preventing accumulation of cruciform DNA intermediates that tether sister chromatids and can undergo nucleolytic processing. We conclude that sister forks can be uncoupled during replication and that, after DSB-induced fork termination, replication is rescued by dormant origin firing or adjacent replicons. We have uncovered a Tel1/ATM- and Mre11-dependent response controlling terminal fork integrity. Our findings have implications for those genome instability syndromes that accumulate DNA breaks during S phase and for forks encountering eroding telomeres

The COMPASS subunit Spp1 protects nascent DNA at the Tus/Ter replication fork barrier by limiting DNA availability to nucleases

Abstract Homologous recombination factors play a crucial role in protecting nascent DNA during DNA replication, but the role of chromatin in this process is largely unknown. Here, we used the bacterial Tus/Ter barrier known to induce a site-specific replication fork stalling in S. cerevisiae. We report that the Set1C subunit Spp1 is recruited behind the stalled replication fork independently of its interaction with Set1. Spp1 chromatin recruitment depends on the interaction of its PHD domain with H3K4me3 parental histones deposited behind the stalled fork. Its recruitment prevents the accumulation of ssDNA at the stalled fork by restricting the access of Exo1. We further show that deleting SPP1 increases the mutation rate upstream of the barrier favoring the accumulation of microdeletions. Finally, we report that Spp1 protects nascent DNA at the Tus/Ter stalled replication fork. We propose that Spp1 limits the remodeling of the fork, which ultimately limits nascent DNA availability to nucleases

Telomere damage induces internal loops that generate telomeric circles

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

Top1- and Top2-mediated topological transitions at replication forks ensure fork progression and stability and prevent DNA damage checkpoint activation

DNA topoisomerases solve topological problems during chromosome metabolism. We investigated where and when Top1 and Top2 are recruited on replicating chromosomes and how their inactivation affects fork integrity and DNA damage checkpoint activation. We show that, in the context of replicating chromatin, Top1 and Top2 act within a 600-base-pair (bp) region spanning the moving forks. Top2 exhibits additional S-phase clusters at specific intergenic loci, mostly containing promoters. TOP1 ablation does not affect fork progression and stability and does not cause activation of the Rad53 checkpoint kinase. top2 mutants accumulate sister chromatid junctions in S phase without affecting fork progression and activate Rad53 at the M–G1 transition. top1 top2 double mutants exhibit fork block and processing and phosphorylation of Rad53 and γH2A in S phase. The exonuclease Exo1 influences fork processing and DNA damage checkpoint activation in top1 top2 mutants. Our data are consistent with a coordinated action of Top1 and Top2 in counteracting the accumulation of torsional stress and sister chromatid entanglement at replication forks, thus preventing the diffusion of topological changes along large chromosomal regions. A failure in resolving fork-related topological constrains during S phase may therefore result in abnormal chromosome transitions, DNA damage checkpoint activation, and chromosome breakage during segregation

Enrichment of 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, 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. Author summary Centromeres are the portions of the chromosomes required for the correct partitioning of genetic material into the daughter cells. In humans, centromeric DNA is made of highly repetitive DNA sequences that hindered its precise molecular characterization until very recently with the development of pivotal technological advances. However, these approaches require the analysis of the whole human genome, while centromeres only represent less than 5%. For this reason, detailed characterization of human centromeres is still very expensive in terms of cost, timing and data analysis. We propose a method called CenRICH that allows to enrich and purify for human centromeric DNA. We prove that this method provides several advantages: 1) it drastically reduces the cost of centromere sequencing; 2) it can be used to study the epigenetic status of centromeres with high level of resolution; 3) it is suitable for single molecule visualization with advanced microscopy techniques. Therefore, CenRICH is a powerful tool to facilitate many future studies in the ever-expanding field of centromere biology, with potential application in study of genetic disease

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

Chromosome instability (CIN) is the most common form of genome instability and is a hallmark of cancer. CIN invariably leads to aneuploidy, a state of karyotype imbalance. Here, we show that aneuploidy can also trigger CIN. We found that aneuploid cells experience DNA replication stress in their first S-phase and precipitate in a state of continuous CIN. This generates a repertoire of genetically diverse cells with structural chromosomal abnormalities that can either continue proliferating or stop dividing. Cycling aneuploid cells display lower karyotype complexity compared to the arrested ones and increased expression of DNA repair signatures. Interestingly, the same signatures are upregulated in highly-proliferative cancer cells, which might enable them to proliferate despite the disadvantage conferred by aneuploidy-induced CIN. Altogether, our study reveals the short-term origins of CIN following aneuploidy and indicates the aneuploid state of cancer cells as a point mutation-independent source of genome instability, providing an explanation for aneuploidy occurrence in tumors