High-resolution physical and functional mapping of the template adjacent DNA binding site in catalytically active telomerase
| Autor: | Haim Manor, Nava Baran, Erez Romi, Michael Shmoish, Marina Gantman, Bosun Min, Kathleen Collins |
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| Rok vydání: | 2007 |
| Předmět: |
Telomerase
Protein subunit Biology Catalysis Tetrahymena thermophila chemistry.chemical_compound Animals Amino Acids Binding site Binding Sites Multidisciplinary Base Sequence Active site Templates Genetic DNA Protozoan Biological Sciences Reverse transcriptase DNA binding site chemistry Biochemistry Mutation biology.protein Primer (molecular biology) DNA Protein Binding |
| Zdroj: | Proceedings of the National Academy of Sciences. 104:8791-8796 |
| ISSN: | 1091-6490 0027-8424 |
| Popis: | 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 K 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. |
| Databáze: | OpenAIRE |
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