טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentKertesz Rosenfeld Karin
SubjectMapping of DNA Binding Sites in Tetrahymena Telomerase
Holoenzyme Proteins by Mass Spectrometry
DepartmentDepartment of Biology
Supervisors Professor Haim Manor
Professor Yoav Arava
Full Thesis textFull thesis text - English Version


Abstract

The telomeric DNA in most eukaryotes consists of short (6-8 nucleotides) G-rich repeats and ends with a single-stranded 3’overhang. Telomerase is a cellular reverse transcriptase that catalyzes elongation of this overhang. The telomerase complex consists of two core components: (i) a catalytic protein subunit designated telomerase reverse transcriptase (TERT). (ii) an RNA molecule designated telomerase RNA (TER) which includes a short sequence serving as a template for synthesis of the telomeric DNA repeats. The telomerase holoenzyme includes, besides the core, accessory proteins which are essential for its assembly and enzymatic activity in vivo.

We have used the Tetrahymena telomerase as a model system for mechanistic studies of the enzyme. Our specific goal was to identify and map the interaction sites of the telomerase holoenzyme with telomeric DNA substrates in active enzyme complexes by site-specific UV cross-linking and mass spectrometry.

In one series of experiments, an oligodeoxyribonucleotide substituted with a 5-Iododeoxyuridine residue, or a 4-Thio-deoxythymidine residue, was cross-linked to protein components of the telomerase by irradiation with ultraviolet Light Emitting Diodes (LEDs). The DNA was subsequently extended by the cross-linked enzyme with a radioactively labeled or unlabeled nucleotide and the complexes were resolved by SDS-polyacrylamide gel electrophoresis. Proteins were then purified from strips cut from the unlabeled gels corresponding to bands observed in the radioactive gels. Mass spectrometric analysis of these proteins revealed a major cross-link site in TERT. Serendipitous cleavage of TERT near the amino acid No. 254 indicated that this site maps within the N-terminal cleavage product, which is located outside the active site of the enzyme. 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 another series of experiments, unresolved cross-linked telomerase-DNA complexes were digested with trypsin, two exonucleases and alkaline phosphatase. Tandem mass spectrometry was then used to search for peptides linked to the residual deoxynucleoside. Using this approach, we identified the phenylalanine residue F351 in the accessory protein p45 as a DNA cross-linking site. Our study constitutes the first direct mapping of DNA interaction sites in telomerase holoenzyme complexes. Moreover, in the course of our research, we have made three significant methodological contributions: First, we built a novel apparatus for UV irradiation. Secondly, we improved the enzymatic digestion protocol for cleavage of ssDNA cross-linked to peptides. Thirdly, our work contributed to the understanding of the reaction leading to cross-linking of Thio substituted nucleotides to proteins.