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High-Resolution Physical and Functional Mapping of the Template Adjacent DNA Binding Site in Catalytically Active Telomerase

Erez Romi, Nava Baran, Marina Gantman, Michael Shmoish, Bosun Min, Kathleen Collins and Haim Manor
Proceedings of the National Academy of Sciences of the United States of America
Vol. 104, No. 21 (May 22, 2007), pp. 8791-8796
Stable URL: http://www.jstor.org/stable/25427752
Page Count: 6
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Since scans are not currently available to screen readers, please contact JSTOR User Support for access. We'll provide a PDF copy for your screen reader.
High-Resolution Physical and Functional Mapping of the Template Adjacent DNA Binding Site in Catalytically Active Telomerase
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Abstract

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 ${\rm K}_{{\rm m}}$ 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.

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