Telomerase is a ribonucleoprotein complex that synthesizes the G-rich DNA found at the 3-ends of linear chromosomes. Human telomerase consists minimally of a catalytic protein (hTERT) and a template-containing RNA (hTR), although other proteins are involved in regulating telomerase activity in vivo. Several chaperone proteins, including hsp90 and p23, have demonstrable roles in establishing telomerase activity both in vitro and in vivo, and previous reports indicate that hsp90 and p23 are required for the reconstitution of telomerase activity from recombinant hTERT and hTR. Here we report that hTERT and hTR associate in the absence of a functional hsp90-p23 heterocomplex. We also report that hsp90 inhibitors geldanamycin and novobiocin inhibit recombinant telomerase even after telomerase is assembled. Inhibition by geldanamycin could be overcome by allowing telomerase to first bind its primer, suggesting a role for hsp90 in loading telomerase onto the telomere. Inhibition by novobiocin could not similarly be overcome but instead resulted in destabilization of the hTERT polypeptide. We propose that the hsp90-p23 complex fine tunes and stabilizes a functional telomerase structure, allowing primer loading and extension.Telomerase is an RNA-dependent DNA polymerase that extends the 3Ј-ends of linear chromosomes, allowing replicating cells to overcome the end replication problem (1). The complete subunit composition of the human telomerase ribonucleoprotein (RNP) 4 complex has yet to be fully elucidated, but the minimally active enzyme requires the reverse transcriptase catalytic subunit (hTERT) and the RNA subunit (hTR), which contains the template (2-5). Recent findings revealed that chaperone proteins, such as hsp90, p23, hsp70, p60, and hsp40/ydj, are also part of the telomerase RNP, at least during part of the cell cycle (6, 7). In fact, these chaperones are required to obtain a functionally active telomerase RNP in vitro, although their precise role in telomerase assembly remains enigmatic (6, 7). hsp90 is a highly conserved and abundant protein found in all eukaryotic cells (8, 9). hsp90 functions as part of a "foldosome" complex that together with other chaperones facilitates the accurate arrangement of numerous proteins (10, 11). This activity is dependent on a number of previously identified functional domains. Important for pharmacological concerns are the N-terminal ATP-binding site (12, 13) and a putative C-terminal ATP-binding site (14, 15). These sites are essential for hsp90 function, because ATP binding and hydrolysis are crucial in the conformational regulation of hsp90 and therefore its effects on client proteins (16). It has been suggested that the two ATP-binding sites act cooperatively, allowing cross-talk mediated by a central charged domain between the two termini (15). hsp90 is a functional homodimer with one homodimerization domain in the C terminus (17) and a second located within the N terminus (16). There are also a number of co-chaperone binding domains, including one for the acidic phos...

No abstract