Shilagardy Khurts

In normal human cells, telomeres shorten with successive rounds of cell division, and immortalization correlates with stabilization of telomere length. These observations suggest that human cancer cells achieve immortalization in large part through the illegitimate activation of telomerase expression. Here, we demonstrate that the rate-limiting telomerase catalytic subunit hTERT is expressed in cycling primary presenescent human fibroblasts, previously believed to lack hTERT expression and telomerase activity. Disruption of telomerase activity in normal human cells slows cell proliferation, restricts cell lifespan, and alters the maintenance of the 3′ single-stranded telomeric overhang without changing the rate of overall telomere shortening. Together, these observations support the view that telomerase and telomere structure are dynamically regulated in normal human cells and that telomere length alone is unlikely to trigger entry into replicative senescence.

Human telomerase reverse transcriptase (hTERT), the catalytic subunit of telomerase, contains motifs conserved among reverse transcriptases. Several nucleic acid-dependent polymerases that share a "fingers, palm, and thumb substructure" were shown to oligomerize. Here we demonstrate that hTERT also has this ability using partially purified recombinant hTERTs and mammalian cells co-expressing differently tagged hTERTs. Human template RNA (hTR), by contrast, has no effect on the structural oligomerization of hTERTs. Therefore, hTERT has an intrinsic ability of oligomerization in the absence of hTR. We identified two separate regions as essential for the oligomerization. The regions, amino acids 301-538 (amino-terminal region) and amino acids 914-928 (carboxyl-terminal region), are outside the fingers and palm substructure covering motif T to D and interact with each other in vivo. A substituted mutant of hTERT, hTERT-D712A-V713I, which was reported as a dominant negative form of hTERT, bound to the wild-type hTERT and inhibited its telomerase activity transiently expressed in telomerase-negative finite normal human fibroblast. The truncated forms of hTERT containing the binding region to the wild-type hTERT partially inhibited the telomerase activity, probably by preventing the wild-type hTERT from forming an oligomer. Taken together, the oligomerization of hTERT is an important step for telomerase activity.