Shortening of the ends of chromosomes limits a cell's lifespan. Some cancer cells avoid this fate through a mechanism called alternative lengthening of telomeres, molecular details of which have now been defined. See Article p.54
During cell division, the genome is duplicated by DNA polymerase enzymes. However, each chromosome's ends are incompletely replicated during duplication, because DNA polymerases require an RNA primer 5′ to the region being synthesized. This means that the repetitive DNA sequences called telomeres that cap the ends of chromosomes shorten at each division, and this shortening limits the replicative lifespans of most cells. During cancer development, cells acquire the ability to divide indefinitely by circumventing telomere shortening — either by upregulating the enzyme telomerase, which extends telomeres, or by activating a mechanism termed alternative lengthening of telomeres (ALT), which is based on a common method of DNA repair, homologous recombination. On page 54, Dilley et al.1 reveal a mechanism that underlies ALT and identify an unusual DNA polymerase that mediates this process.
During homologous recombination, a double-strand break in the DNA of one chromosome is repaired by a DNA polymerase using template DNA that is taken from a matching sister chromatid — an identical DNA molecule generated during replication. Cancer cells that use ALT often show higher levels of DNA damage at telomeres than do non-ALT cells2, which may predispose them to use homologous recombination to repair breaks in telomeric DNA. In human ALT cancer-cell lines, evidence of enhanced homologous recombination at telomeres includes an increase in telomere exchanges between sister chromatids compared to other cell lines3, and evidence for the copying of DNA tags from one chromosome end to another4. An estimated 10–15% of tumours use ALT to maintain their telomeres, making this process an important target for cancer therapy5,6. However, dissection of the molecular mechanisms that underlie ALT has been challenging.
There are two ways in which telomeres might use homologous recombination to maintain their length. In the first model, unequal exchange of DNA between sister telomeres creates a longer and a shorter telomere, one of which is inherited by each daughter cell. The cells that inherit longer telomeres eventually outcompete those that have shorter telomeres. In the second model, which is increasingly gaining favour, telomeric DNA is synthesized using an existing piece of telomeric template, either from another telomere or from free molecules of repetitive DNA called extrachromosomal telomeric DNA that are found in ALT cells5.
Dilley et al. provide strong support for the second model. To encourage homologous recombination in ALT cells, the authors exploit a system they had previously engineered7 to create targeted double-strand breaks in telomeres by fusing the Fok1 nuclease enzyme, which cleaves DNA, to the telomere-binding protein TRF1. They observe a tenfold increase in telomeric DNA synthesis after TRF1–Fok1 induction in cells known to use ALT. Furthermore, they show that this synthesis is unidirectional and processive — capable of synthesizing long tracts of telomere repeats typically 20 kilobases long (the length of an average ALT telomere). The kinetics of this synthesis are consistent with those that could cause large fluctuations in telomere length, as is seen in ALT cells8.
The characteristics and kinetics of this DNA synthesis match those of a phenomenon called break-induced replication, which is a telomere-maintenance mechanism in yeast strains that lack telomerase9. Break-induced replication is a form of homologous recombination that initiates DNA replication when only one end of a double-strand break shares sequence similarity with a template. Dilley et al. term this process in mammalian ALT cells break-induced telomere synthesis.
The authors next set out to characterize the proteins responsible for break-induced telomere synthesis. The protein Rad51 has a key role in homologous recombination, and is required for break-induced replication in yeast9. But, surprisingly, Dilley and colleagues found that Rad51 was dispensable for break-induced telomere synthesis in ALT cells. Rather, a complex that consists of the polymerase POLδ and the proteins PCNA and RFC1-5 is found at sites of DNA damage in ALT cells and is required for break-induced telomere synthesis (Fig. 1). The authors theorize that this atypical complex is responsible for the dominant pathway of telomere synthesis in ALT cells.
Although Dilley et al. shed light on the mechanisms underlying ALT in cancer cells, their findings also open up new questions. For instance, the authors demonstrated that they could trigger break-induced telomere synthesis in both ALT and telomerase-producing cells, so why is this method of telomere replication not operative in most cancer cells? It is unclear what induces the ALT mechanism and how that mechanism is specifically sustained in the 10–15% of cancer cell types that use ALT. The authors provide one possible explanation — that ALT cells have higher rates of persistent telomere damage than other cancer cells.
Alternatively, it might be that there is a change in the way in which telomeric DNA is packaged around histone proteins to form chromatin. Disruption of histone function has been shown10 to induce ALT-like characteristics in cells, suggesting a mechanistic link between altered telomere histones and the ALT mechanism. Moreover, mutations in a chromatin-remodelling protein complex, ATRX–DAXX, are highly recurrent in human ALT tumours11,12,13,14. The current work does not address how mutations in the ATRX–DAXX complex lead to ALT, but this will be an interesting avenue for further investigation.
Dilley and colleagues' link between break-induced telomere synthesis and ALT provides insights that might help us to further understand how ALT is initiated and maintained in human cancer cells. In the future, a more in-depth understanding of these processes might lead to the development of therapies targeting human cancers that depend on ALT.