RECONSTITUTION OF HUMAN TELOMERASE WITH THE TEMPLATE RNA COMPONENT HTR AND THE CATALYTIC PROTEIN SUBUNIT HTRT

The maintenance of chromosome termini, or telomeres, requires the acti on of the enzyme telomerase, as conventional DNA polymerases cannot fu lly replicate the ends of linear molecules(1-4). Telomerase is express ed and telomere length is maintained in human germ cells and the great majority of primary human tumours(5,6). However, telomerase is not de tectable in most normal somatic cells; this corresponds to the gradual telomere loss observed with each cell division(7-9). It has been prop osed that telomere erosion eventually signals entry into senescence or cell crisis and that activation of telomerase is usually required for immortal cell proliferation(10). In addition to the human telomerase RNA component (hTR; ref, 11), TP1/TLP1 (refs 12,13), a protein that is homologous to the p80 protein associated with the Tetrahymena enzyme( 14), has been identified in humans. More recently, the human telomeras e reverse transcriptase (hTRT; refs 15, 16), which is homologous to th e reverse transcriptase (RT)like proteins associated with the Euplotes aediculatus (Ea_p123), Saccharomyces cerevisiae (Est2p) and Schizosac charomyces pombe (SpTrt1) telomerases(15,17), has been reported to be a telomerase protein subunit. A catalytic function has been demonstrat ed for Est2p in the RT-like class but not for p80 or its homologue(17, 18). We now report that in vitro transcription and translation of hTRT when co-synthesized or mixed with hTR reconstitutes telomerase activi ty that exhibits enzymatic properties like those of the native enzyme, Single amino-acid changes in conserved telomerase-specific and RT mot ifs reduce or abolish activity, providing direct evidence that hTRT is the catalytic protein component of telomerase. Normal human diploid c ells transiently expressing hTRT possessed telomerase activity, demons trating that hTRT is the limiting component necessary for restoration of telomerase activity in these cells. The ability to reconstitute tel omerase permits further analysis of its biochemical and biological rol es in cell aging and carcinogenesis.