De novo telomere addition at chromosome breaks: Dangerous Liaisons

2243 The Rockefeller University Press J. Cell Biol. Vol. 216 No. 8 2243–2245 https://doi.org/10.1083/jcb.201705156 All eukaryotic cells with linear chromosomes face the problem of terminal DNA sequence loss that occurs as a result of either incomplete replication of the DNA strand synthesized by the lagging strand replication machinery or accidental collapse of the replication forks. If left unrepaired, these losses will eventually trigger the DNA damage response (DDR) and cell cycle arrest. Most eukaryotes use the enzyme telomerase that specializes in supplementing lost sequences at the chromosome ends to counteract this problem. Telomerase is minimally composed of the catalytic subunit, a telomerase reverse transcription, and the RNA component that serves as a scaffold for telomerase subunit assembly and also carries a region that templates the synthesis of telomeric DNA repeats. The template region of telomerase RNA determines both the guanine-rich sequence of telomeric repeats and the specificity of telomerase for chromosome ends because the template region has to anneal to the single-strand DNA (ssDNA) tail exposed at the site of its action. To further reinforce the specificity of telomerase for chromosome ends, a telomerase recruitment mechanism has evolved that relies on specialized proteins that bind telomeric ssDNA with high affinity and sequence specificity such as Cdc13 in budding yeast and POT1 in mammalian cells. However, in spite of these adaptations, telomerase does interfere with repair of DNA double strand breaks (DSBs) and may occasionally add telomeric repeats to either spontaneous or induced DSBs, a process known as chromosome healing by de novo telomere addition (Pennaneach et al., 2006). In this issue, Ouenzar et al. describe a novel mechanism that restricts the action of telomerase by spatial exclusion from sites of DNA repair. Although de novo telomere addition at internal non-telomeric sites stabilizes the end of a broken chromosome, it leads to the loss of large portions of chromosome arms that usually endangers cell viability. Previous work in budding yeast identified molecular mechanisms responsible for curbing the action of telomerase on DSBs and uncovered that these mechanisms are intimately linked to DDR signaling (Makovets and Blackburn, 2009; Zhang and Durocher, 2010). Upon detection of DSBs, Mec1 kinase, a budding yeast orthologue of ATR, initiates a signaling cascade by phosphorylating multiple targets. One of these targets phosphorylated in a MEC1-RAD53-DUN1– dependent manner is a Pif1 helicase, a known telomerase inhibitor that dislodges telomerase from its DNA substrate (Boulé et al., 2005). Importantly, this phosphorylation of Pif1 specifically mediates telomerase inhibition at DNA breaks, but not at telomeres (Makovets and Blackburn, 2009). In addition, activated Mec1 that accumulates at resected DNA ends directly phosphorylates Cdc13 on Ser 306, and this phosphorylation event prevents accumulation of Cdc13 at DNA ends with very short telomere-like sequence seeds, thereby suppressing telomere addition to accidental DSBs (Zhang and Durocher, 2010). Although these sophisticated mechanisms elegantly incorporated into the DDR diminish the action of telomerase on DSBs, it appears to not be the whole story. Indeed, Ouenzar et al. (2017) add a new layer of regulation to previous results by showing that telomerase action at DNA breaks is restricted by the sequestration of its RNA component in the nucleolus. Ouenzar et al. (2017) have used FISH to quantify intracellular distribution of TLC1 RNA, a telomerase RNA in budding yeast, at different phases of the cell cycle. They found that individual telomerase RNA molecules formed foci in the nucleoplasm during the G1 and S phases of the cell cycle, but became sequestered in the nucleoli in the G2/M phase. Interestingly, the cell cycle–dependent trafficking of TLC1 RNA into the nucleoli required Pif1 activity in the nucleoplasm, which removes telomerase from telomeres in late S/G2. The majority of DSBs in budding yeast are repaired by homologous recombination (HR), which is active in G2 phase and occurs exclusively in the nucleoplasm, suggesting that nucleolar localization of TLC1 RNA could serve to reduce the interference of telomerase with HR in DSB repair. To interrogate a functional significance of nucleolar localization of TLC1 RNA in the context of DSB repair, Ouenzar et al. (2017) examined the distribution of TLC1 RNA in the cells treated with bleomycin, a radiomimetic drug that generates DSBs. In bleomycin-treated cells in the G2/M phase, TLC1 RNA redistributed slightly into the nucleoplasm, indicating that its trafficking between nucleoplasm and nucleolus is affected Telomerase counteracts the loss of terminal DNA sequences from chromosome ends; however, it may erroneously add telomeric repeats to DNA double-strand breaks. In this issue, Ouenzar et al. (2017. J. Cell Biol. https ://doi .org /10 .1083 /jcb .201610071) uncover cell cycle–dependent sequestration of the telomerase RNA in nucleoli, a process that excludes telomerase from DNA repair sites. De novo telomere addition at chromosome breaks: Dangerous Liaisons