One of the first steps in studying the enzyme telomerase was the generation of telomerase mutant model organisms. In 1993, a research team, Victoria Lundblad and Elizabeth Blackburn studied a telomerase-null yeast strain (4). Amazingly, although most of the cells of this yeast strain did not survive, some were able to maintain the length of their telomeres without the enzyme telomerase (1, 4). How was this even possible? Recent research has uncovered a second, rarely-seen mechanism of telomere maintenance known as the Alternative Lengthening of Telomeres (ALT) pathway. Typically occurring in cells lacking telomerase activity, the ALT pathway provides a means of replicating telomeres through homologous recombination, and is present in roughly 15 percent of cancers (1). In this section we will explore the characteristics of ALT-positive cells, current models of ALT function, and what factors cause it to occur.
Characteristics of the ALT pathway and PML bodies
Although ALT has never been observed in normal cells, it has been identified in a number of cancers, immortalized cell lines, and transgenic organisms (1). Its phenotype is unique. ALT-positive cells retain many qualities of normal telomeres: the presence of the shelterin complex, TTAGGG repeats, a 3’ G-strand overhang, and the ability to form T-loops (1). However, there are a number of aberrant structures present, most notably large amounts of extrachromosomal telomeric DNA. This abnormal telomeric DNA comes in many forms: T-circles (circular double stranded telomeric DNA), C/G-circles (partially single stranded DNA representative of the C or G rich strands, respectively), linear double-stranded DNA, or T-complex DNA (highly branched, abnormal DNA aggregates) (1). C-circles are regarded as the best indicators of the presence of the ALT, and the number of C-circles appears to be closely correlated with ALT activity (1).
PML bodies are aggregates of promyelocytic leukemia (PML) protein with other nuclear proteins which are present within many cell types, including both telomerase-positive and ALT cell lines. Although remaining functionally unknown, constituents of PML bodies include factors functioning in tumour formation, cellular senescence, stress response and DNA repair, suggesting that the aggregates may be functionally diverse (3). ALT-associated PML bodies are specific to ALT cell lines and contain a number of unique factors including telomeric DNA, and are a key biomarker of ALT cell lines. The diverse functional potential of PML bodies has lead to suggestions that they may play a role in homologous recombination within ALT cell lines, utilizing telomeric DNA and recombination within the aggregates (3).
Unequal telomere sister chromatid exchange (Unequal T-SCE) model
Sister chromatid exchange is a process by which the sister chromatids swap equivalent segments of genetic material through homologous recombination (1). Typically observed during DNA repair of broken replication forks, it was noted that sister chromatid exchange of the telomere regions (telomere sister chromatid exchange, or T-SCE) occurred significantly more frequently in ALT-positive cells (1). This led to the hypothesis that unequal recombination was occurring. This model suggests that a very small portion of one telomere is swapped for a significantly larger portion of the other telomere, resulting in one chromatid with a longer telomere and the other possessing a shortened telomere (1). However, this model relies on the potentially troubling assumption that all of the long telomeric chromosomes will segregate to one daughter cell, and all of the short telomeric chromosomes will move to the other cell. The cell with the long chromatids would be able to undergo more replications than its counterpart (1). Currently, there is a lack of evidence to support this model, although recent evidence has emerged that sister chromatids segregate non-randomly in a subset of mouse cells (1).
Homologous recombination-dependent DNA replication (HR) model
This is currently the most accepted model of ALT function. The homologous recombination-dependent (HR-dependent) DNA replication model was based off the observation that a DNA tag inserted into the telomeres of ALT-positive cells was copied from one telomere to the other. This meant that the number of tagged telomeres actually increased (1). In this model, a single stranded telomeric end invades another telomere’s double strand, using the double strand as a template for DNA replication (1). This results in a net gain of telomeric DNA. The HR-dependent model also allows the various varieties of extrachromosomal DNA to act as templates for telomere replication; sister chromatids do not necessarily need to be used (1). This model is favored over the unequal T-SCE model because it explains how the number of tagged telomeres can increase (1). In the unequal T-SCE model, since tagged telomeres are merely exchanged, their numbers do not increase. Additionally, this model explains how the DNA sequence of telomeres can change depending on which region of double stranded DNA is used as a template.
Promoters of ALT: The MRN complex and creation of the 3’ overhang
The MRN complex (comprised of the proteins MRE11A, RAD50, and NBS1) was the first component shown to be necessary for ALT-mediated telomere lengthening to occur (1). This complex is a DNA damage sensor that recruits the ataxia telangiectasia mutated (ATM) protein (a master controller of cell-cycle signaling pathways) to a double strand break point. ATM then creates the long 3’ overhangs necessary for the strand invasion proposed in the homologous recombination-dependent ALT model (1).
Inhibitors of ALT: p53, ATRX/DAXX/H3F3A, and the shelterin complex
One gene thought to inhibit the ALT pathway is the tumor supressor gene p53. p53 is mutated or inactive in 95 percent of ALT-positive immortalized cell lines (2). It is a protein with a variety of cell functions: binding DNA and activating signaling pathways, triggering transcription of target genes, altering the cell-cycle, and activating DNA repair machinery (2). A p53 mutant that has lost the ability to trigger DNA transcription remains able to suppress homologous recombination, inhibiting cell proliferation in p53-deficient ALT-positive cell lines (2). This suggests that p53 plays a significant role in preventing homologous recombination-dependent ALT (2). For more information about the tumor supressor gene p53, see Chapter 6: Tumor suppressor genes.
In addition to p53, ATRX and DAXX proteins also inhibit the ALT pathway. The ATRX and DAXX proteins form a heterodimeric complex responsible for remodeling chromatin (including telomeric DNA) during S-phase (2). The ATRX-DAXX complex is required for deposition of H3.3, a histone associated with open chromatin, transcription factor binding regions, and telomeres (2). H3F3A is the gene that encodes H3.3. Mutations in any of the genes encoding these proteins limits H3.3 incorporation into chromatin, disrupting telomeres and facilitating their recombination.
Finally, the shelterin complex is also hypothesized to inhibit the ALT pathway (1). Two components of the ALT pathway: POT1 and TRF2 exhibit anti-ALT properties in vivo (1). TRF2 promotes the formation of T-loops and protects against enzymatic damage, indicating that it suppresses recombination. POT1 appears to regulate replication of the 3’ G-strand, so its inhibition may increase the number of broken replication forks and T-SCE events (1). The shelterin complex binds not only to normal telomeres, but also extrachromosomal telomeric DNA (1). It is thought that the increased amount of telomeric DNA in ALT-positive cells results in a relative deficiency of the shelterin complex (it is not upregulated in response) (1). This may mean that the shelterin complex protects a smaller portion of the telomeres in an ALT-positive cell, resulting in an increase in recombination events (1). For more information about the shelterin complex, see the Telomerase section.
1. Cesare, A. J., & Reddel, R. R. (2010). Alternative lengthening of telomeres: models, mechanisms and implications. Nature reviews. Genetics, 11(5), 319–30. doi:10.1038/nrg2763
2, Gocha, A. R. S., Harris, J., & Groden, J. (2012). Alternative mechanisms of telomere lengthening: Permissive mutations, DNA repair proteins and tumorigenic progression. Mutation research, 1–9. doi:10.1016/j.mrfmmm.2012.11.006
3. Yeager TR, Neumann AA, Englezou A, Huschtscha LI, Noble JR, Reddel R. Telomerase-negative immortalized human cells contain a novel type of promyelocytic leukemia (PML) body. Cancer Res. 1999;59:4175-4179.
4. Lundblad V., Blackburn E.H. (1993). An alternative pathway for yeast telomere maintenence rescues est1- senescence. Cell, 73, 347-360.