title: High-resolution physical and functional mapping of the template adjacent DNA binding site in catalytically active telomerase.
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 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.
authors.affiliation: Department of Biology, Technion-Israel Institute of Technology, Haifa 32000, Israel.
authors.forename: Erez
authors.initials: E
authors.lastname: Romi
authors.forename: Nava
authors.initials: N
authors.lastname: Baran
authors.forename: Marina
authors.initials: M
authors.lastname: Gantman
authors.forename: Michael
authors.initials: M
authors.lastname: Shmoish
authors.forename: Bosun
authors.initials: B
authors.lastname: Min
authors.forename: Kathleen
authors.initials: K
authors.lastname: Collins
authors.forename: Haim
authors.initials: H
authors.lastname: Manor
mesh_terms: D000596:Amino Acids / Q000235:genetics / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D016054:DNA, Protozoan / Q000378:metabolism*; D009154:Mutation / Q000235:genetics; D011485:Protein Binding; D019098:Telomerase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D013698:Templates, Genetic; D016808:Tetrahymena thermophila / Q000235:genetics* / Q000378:metabolism*
chemical_list: D000596:Amino Acids; D016054:DNA, Protozoan; D019098:Telomerase