Telomeres are composed of simple tandem DNA repeats that protect the ends of linear chromosomes from replicative erosion or inappropriate DNA damage response mechanisms. The mammalian Protection Of Telomeres (POT1) protein interacts with single-stranded telomeric DNA and can exert positive and negative effects on telomere length. Of four distinct POT1 homologs in the roundworm Caenorhabditis elegans, deficiency for POT-1 or POT-2 resulted in progressive telomere elongation that occurred because both proteins negatively regulate telomerase. We created a POT-1::mCherry fusion protein that forms discrete foci at C. elegans telomeres, independent of POT-2, allowing for live analysis of telomere dynamics. Transgenic pot-1::mCherry repressed telomerase in pot-1 mutants. Animals deficient for pot-1, but not pot-2, displayed mildly enhanced telomere erosion rates in the absence of the telomerase reverse transcriptase, trt-1. However, trt-1; pot-1 double mutants exhibited delayed senescence in comparison to trt-1 animals, and senescence was further delayed in trt-1; pot-2; pot-1 triple mutants, some of which survived robustly in the absence of telomerase. Our results indicate that POT-1 and POT-2 play independent roles in suppressing a telomerase-independent telomere maintenance pathway but may function together to repress telomerase.

Canonical telomere repeats at chromosome termini can be maintained by a telomerase-independent pathway termed alternative lengthening of telomeres (ALT). Human cancers that survive via ALT can exhibit long and heterogeneous telomeres, although many telomerase-negative tumors possess telomeres of normal length. Here, we report that Caenorhabditis elegans telomerase mutants that survived via ALT possessed either long or normal telomere lengths. Most ALT strains displayed end-to-end chromosome fusions, suggesting that critical telomere shortening occurred before or concomitant with ALT. ALT required the 9-1-1 DNA damage response complex and its clamp loader, HPR-17. Deficiency for the POT-2 telomere binding protein promoted ALT in telomerase mutants, overcame the requirement for the 9-1-1 complex in ALT, and promoted ALT with normal telomere lengths. We propose that telomerase-deficient human tumors with normal telomere lengths could represent a mode of ALT that is facilitated by telomere capping protein dysfunction. PROTECTION OF TELOMERES-2 | telomere maintenanceT elomeres are tandem repeat tracts that cap the ends of linear chromosomes and erode with each cell cycle because of the inability of canonical DNA polymerases to completely replicate chromosome termini (1). However, the ribonucleoprotein telomerase combats this erosion by adding de novo telomeric repeats via reverse transcription (2). Progressive telomere shortening during proliferation of human somatic cells can trigger senescence, which serves as a major tumor suppression mechanism (3). Bypass of senescence and further cell proliferation leads to critical telomere shortening and crisis, resulting in high levels of cell death due to chromosome instability (4). Many tumors overcome this proliferation barrier by activating expression of the telomerase reverse transcriptase. The remaining 10-15% of human tumors do not possess telomerase activity (5, 6).S. cerevisiae strains deficient for telomerase can survive with long and heterogeneous telomeres composed of amplified telomere repeats (type II survivors) (7,8). Analogous to type II survivors, long and heterogeneous telomeres occur in almost all in vitro immortalized cell lines and tumor-derived cells lines that are telomerase-negative (9-11). Furthermore, extensive analysis of telomerase activity and telomere length from primary human tumor samples has revealed that certain telomerase-negative tumors possess long and heterogeneous telomeres, defined as alternative lengthening of telomeres (ALT) tumors (9). However, the majority (81/108) of primary tumors lacking telomerase activity have telomeres of normal lengths (9,(12)(13)(14)(15). This subset of telomerase-negative tumors may have become neoplastic before exhausting their telomere repeat reserves (9, 16). Alternatively, these tumors could have evolved via the same telomeredriven crisis events that give rise to ALT tumors, followed by activation of a distinct form of ALT that maintains telomeres of apparently normal lengths (9).Homologous recombinatio...