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High-resolution physical and functional mapping of the template adjacent DNA binding site in catalytically active telomerase

Telomerase is a cellular reverse transcriptase, which utilizes an integral RNA template to extend single-stranded telomeric DNA. We used site-specific photocrosslinking to map interactions between DNA primers and the catalytic protein subunit (tTERT) of Tetrahymena thermophila telomerase in function...

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Bibliographic Details
Published in:Proceedings of the National Academy of Sciences - PNAS 2007-05, Vol.104 (21), p.8791-8796
Main Authors: Romi, Erez, Baran, Nava, Gantman, Marina, Shmoish, Michael, Min, Bosun, Collins, Kathleen, Manor, Haim
Format: Article
Language:English
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Summary:Telomerase is a cellular reverse transcriptase, which utilizes an integral RNA template to extend single-stranded telomeric DNA. We used site-specific photocrosslinking to map interactions between DNA primers and the catalytic protein subunit (tTERT) of Tetrahymena thermophila telomerase in functional enzyme complexes. Our assays reveal contact of the single-stranded DNA adjacent to the primer-template hybrid and tTERT residue W187 at the periphery of the N-terminal domain. This contact was detected in complexes with three different registers of template in the active site, suggesting that it is maintained throughout synthesis of a complete telomeric repeat. Substitution of nearby residue Q168, but not W187, alters the Km for primer elongation, implying that it plays a role in the DNA recognition. These findings are the first to directly demonstrate the physical location of TERT-DNA contacts in catalytically active telomerase and to identify amino acid determinants of DNA binding affinity. Our data also suggest a movement of the TERT active site relative to the template-adjacent single-stranded DNA binding site within a cycle of repeat synthesis.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.0703157104