N-terminal domains of the human telomerase catalytic subunit required for enzyme activity in vivo

Most tumor cells depend upon activation of the ribonucleoprotein enzyme tel omerase for telomere maintenance and continual proliferation. The catalytic activity of this enzyme can be reconstituted in vitro with the RNA (hTR) a nd catalytic (hTERT) subunits. However, catalytic activity alone is insuffi cient for the full in vivo function of the enzyme. In addition, the enzyme must localize to the nucleus, recognize chromosome ends, and orchestrate te lomere elongation in a highly regulated fashion. To identify domains of hTE RT involved in these biological functions, we introduced a panel of 90 N-te rminal hTERT substitution mutants into telomerase-negative cells and assaye d the resulting cells for catalytic activity and, as a marker of in vivo fu nction, for cellular proliferation. We found four domains to be essential f or in vitro and in vivo enzyme activity, two of which were required for hTR binding. These domains map to regions defined by sequence alignments and m utational analysis in yeast, indicating that the N terminus has also been f unctionally conserved throughout evolution. Additionally, we discovered a n ovel domain, DAT, that "dissociates activities of telomerase," where mutati ons left the enzyme catalytically active, but was unable to function in viv o. Since mutations in this domain had no measurable effect on hTERT homomul timerization, hTR binding, or nuclear targeting, we propose that this domai n is involved in other aspects of in vivo telomere elongation. The discover y of these domains provides the first step in dissecting the biological fun ctions of human telomerase, with the ultimate goal of targeting this enzyme for the treatment of human cancers.