Polymerization defects within human telomerase are distinct from telomerase RNA and TEP1 binding.
The minimal, active core of human telomerase is postulated to contain two components, the telomerase RNA hTER and the telomerase reverse transcriptase hTERT. The reconstitution of human telomerase activity in vitro has facilitated the identification of sequences within the telomerase RNA and the RT motifs of hTERT that are essential for telomerase activity. However, the precise role of residues outside the RT domain of hTERT is unknown. Here we have delineated several regions within hTERT that are important for telomerase catalysis, primer use, and interaction with the telomerase RNA and the telomerase-associated protein TEP1. In particular, certain deletions of the amino and carboxy terminus of hTERT that retained an interaction with telomerase RNA and TEP1 were nonetheless completely inactive in vitro and in vivo. Furthermore, hTERT truncations lacking the amino terminus that were competent to bind the telomerase RNA were severely compromised for the ability to elongate telomeric and nontelomeric primers. These results suggest that the interaction of telomerase RNA with hTERT can be functionally uncoupled from polymerization, and that there are regions outside the RT domain of hTERT that are critical for telomerase activity and primer use. These results establish that the human telomerase RT possesses unique polymerization determinants that distinguish it from other RTs.
Beattie
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TL
Ontario Cancer Institute/Amgen Institute, Department of Medical Biophysics, University of Toronto, Toronto, Ontario M5G 2C1 Canada.
Zhou
W
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Robinson
M O
MO
Harrington
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D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D020134:Catalytic Domain; D002460:Cell Line; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins; D006367:HeLa Cells; D006801:Humans; D007668:Kidney; D012313:RNA / Q000235:genetics*; D016601:RNA-Binding Proteins; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017384:Sequence Deletion; D019098:Telomerase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D014158:Transcription, Genetic; D014162:Transfection
D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; C104342:TEP1 protein, human; C422952:telomerase RNA; D012313:RNA; D019098:Telomerase