title: Mapping of DNA binding sites in the Tetrahymena telomerase holoenzyme proteins by UV cross-linking and mass spectrometry.
abstract: The Tetrahymena telomerase holoenzyme consists of a major catalytic protein [telomerase reverse transcriptase (TERT)], an RNA subunit, and accessory proteins. We used site-specific UV cross-linking and mass spectrometry to map interactions between the holoenzyme and the telomeric DNA. In one series of experiments, an oligodeoxyribonucleotide containing a 5-iododeoxyuridine residue or 4-thio-deoxythymidine residue was cross-linked to the telomerase by irradiation with UV light-emitting diodes. The DNA was extended by the cross-linked enzyme with a radioactively labeled or unlabeled nucleotide. The complexes were subsequently resolved by SDS-PAGE. Proteins were isolated from strips in the unlabeled gels corresponding to bands observed in the radioactive gels. Mass spectrometric analysis of these proteins revealed a major cross-linking site in TERT. Serendipitous cleavage of TERT near amino acid 254 indicated that this site maps within the N-terminal cleavage product, which includes primarily the telomerase essential N-terminal (TEN) domain. Moreover, the absence of this N-terminal segment in TERT was found to cause a reduction in DNA binding by the telomerase and/or its activity to undetectable levels. In other experiments, similar unresolved cross-linked complexes were digested with trypsin, two exonucleases, and alkaline phosphatase. Tandem mass spectrometry was then used to search for peptides linked to the residual deoxyribonucleoside. Using this approach, we identified the phenylalanine residue F351 in the accessory protein p45 as a minor DNA cross-linking site. Our study constitutes the first direct mapping of DNA interaction sites in telomerase holoenzyme complexes. This mapping represents a significant contribution to the understanding of the mechanism of telomere extension by telomerase.
authors.affiliation: Department of Biology, Technion-Israel Institute of Technology, Haifa 32000, Israel.
authors.forename: Karin Kertesz
authors.initials: KK
authors.lastname: Rosenfeld
authors.forename: Tamar
authors.initials: T
authors.lastname: Ziv
authors.forename: Svetlana
authors.initials: S
authors.lastname: Goldin
authors.forename: Fabian
authors.initials: F
authors.lastname: Glaser
authors.forename: Haim
authors.initials: H
authors.lastname: Manor
mesh_terms: D000595:Amino Acid Sequence; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D016054:DNA, Protozoan / Q000235:genetics / Q000378:metabolism*; D020035:Holoenzymes / Q000235:genetics / Q000378:metabolism*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D011485:Protein Binding; D017386:Sequence Homology, Amino Acid; D019098:Telomerase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016615:Telomere / Q000235:genetics*; D016808:Tetrahymena thermophila / Q000201:enzymology* / Q000235:genetics; D014466:Ultraviolet Rays
chemical_list: D003432:Cross-Linking Reagents; D016054:DNA, Protozoan; D020035:Holoenzymes; D019098:Telomerase