Telomerase misbehaves after a breakup

Cells must distinguish between broken chromosome ends and natural ends, or telomeres. Broken chromosomes require repair, but telomeres need to be protected from repair because joining two telomeres would result in disastrous chromosome fusions. Telomerase is the enzyme that maintains chromosome ends by adding repeated DNA sequences. If directed to a DNA break, telomerase would erroneously create a new telomere, precipitating the loss of essential genetic material distal from the break site. Although extensive research has elucidated how telomeres evade DNA repair pathways, the question of telomerase’s misguided activity at DNA breaks in human cells has remained enigmatic. On page 763 of this issue, Kinzig et al. (1) report that telomerase can cause harmful neotelomere formation in human cells. They also show that such events are kept to a minimum by the naturally low prevalence of telomerase and the intervention of the ataxia telangiectasia and Rad3-related (ATR) cellular signaling pathway.

Telomeres consist of repetitive DNA sequences protected by a protein complex called shelterin. In the absence of telomerase, telomeres shorten with each cell division, eventually reaching a point where they no longer suppress DNA damage signaling, which leads to cellular senescence (2). In the germ line, stem cells, and most cancers, telomerase counters this shortening by adding telomeric repeats to chromosome ends. Telomerase is a ribonucleoprotein complex with reverse transcriptase activity that uses its intrinsic RNA template to elongate telomeres. It is recruited to telomeres by the shelterin component TPP1 and binds to the telomere’s single-stranded end through base pairing with its RNA template (see the figure). Unlike telomeres, a DNA double-strand break (DSB) lacks both TPP1 and telomeric DNA sequences that could prime telomerase. Thus, it has not been clear whether telomerase can add neotelomeres at DNA breaks in humans. Indications of this possibility do exist. Patients with deleted distal chromosome ends have telomeric sequences at the DNA breakpoint. Furthermore, neotelomere formation occurs in other organisms, such as ciliated protozoa (3), and can be promoted by induced DSBs in yeast and mice (4–6).

Figure. Telomerase action at natural telomeres and double-strand breaks.

At natural chromosome ends, single-stranded telomeric DNA repeats () provide a primer for extension by the RNA-protein enzyme telomerase (top right). Most double-strand breaks are repaired, but neotelomere formation can occur with a potentially disastrous loss of distal sequences. How telomerase initiates at nontelomeric DNA () is unknown, but once initiated, the reaction proceeds normally (bottom right).

To address the frequency and mechanism of neotelomere formation in human cells, Kinzig et al. engineered a cell line in which they could produce a DSB by CRISPR-Cas9 or the I-SceI endonuclease. They found that telomerase can add telomeric repeats to DSBs, which are then converted to functional telomeres. Neither the ataxia telangiectasia mutated (ATM) pathway—typically activated in response to DSBs—nor common DNA repair factors prevented this inappropriate action. Rather, Kinzig et al. identified ATR, a DNA damage signaling protein that coordinates the response to replication stress and whose action at natural telomeres is suppressed by the shelterin protein protection of telomeres 1 (POT1) (7).

Although this finding is unexpected, one should consider that in the absence of an insult, such as ionizing radiation, most DSBs are the result of replication stress. Stalled replication forks can reverse, resulting in a structure called a chicken foot. Telomerase acts on such structures when they arise at telomeres (8). Future research is needed to elucidate the prevalence of neotelomere formation at replication-induced DSBs and at chicken foot structures as well as the function of ATR in that context.

There are contexts in which DSBs are intentionally created. During meiosis, the enzyme Spo11 generates DSBs that drive recombination between homologous chromosomes, ensuring correct alignment and contributing to genetic diversity (9). Given that meiotic cells exhibit high telomerase activity and can generate more than 200 DSBs, it is plausible that some may be converted to neotelomeres. The resulting chromosome truncations would often be incompatible with embryonic development. Thus, neotelomere formation at meiotic DSBs could cause early miscarriage. Alternatively, neotelomeres formed close to chromosome ends, resulting only in the loss of nonvital genetic material, would lead to germinal structural variants with terminal deletions. Indeed, Kinzig et al. culled published DNA sequences and found 36 patients harboring terminal chromosome deletions associated with neotelomere formation.

Neotelomere formation may be particularly important in cancer, where telomerase is reactivated in 90% of cases. Chromosome rearrangements can drive cancer by creating oncogenes or increasing their copy number or by eliminating tumor-suppressor genes. However, high instability is not sustainable for cell growth. Kinzig et al. propose that the healing of DSBs by telomerase may allow cancer cells to cope with genome instability.

Some species have adopted telomere capping of programmed DSBs as a developmental mechanism. In ciliated protozoa, a few very large germinal chromosomes are broken, giving rise to a high number of small somatic chromosomes, which are stabilized by neotelomere formation (3). Germline genes in the nematode Ascaris are eliminated during early development through programmed DSBs and telomere healing (10). From an evolutionary standpoint, it is possible that neotelomere formation has contributed to speciation. Indeed, the great apes have many instances of distal deletions or inversions (11), which may have necessitated the action of telomerase on nontelomeric substrates.

It is noteworthy how effective human cells are at suppressing this treacherous activity of telomerase, even in contexts where ATR is suppressed and telomerase activity is high. A potential simple explanation could be the lack of telomeric sequences and TPP1 at DSBs. Telomerase RNA typically pairs with five nucleotides at the telomeric end, and although it can still add repeats with as few as two nucleotides of complementarity (1, 12), these are likely inefficient primers. Similarly, TPP1 aids in telomerase recruitment to telomeres (13). Telomerase is capable of extension without TPP1, but this activity is minimal (14). Future research may reveal whether these factors, combined with the protective role of ATR, are sufficient to prevent telomerase action at DSBs or whether additional safeguarding mechanisms exist. ■