טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentGoldin Svetlana
SubjectTracing the Path of Telomeric DNA in Active Tetrahymena
Telomerase Holoenzyme Complexes: Mapping of DNA
Contact Sites in the RNA Subunit
DepartmentDepartment of Biology
Supervisors Professor Haim Manor
Professor Yoav Arava
Full Thesis textFull thesis text - English Version


Abstract

Telomeres are DNA-protein complexes found at the end of linear eukaryotic chromosomes. The telomeric complexes protect chromosomes from degradation, recombination, and fusion with other chromosomes. In most eukaryotes, the telomeric DNA consists of short sequence repeats ending in a single-stranded G-rich 3’-overhang. Telomerase is a cellular reverse transcriptase that catalyzes the extension of the telomeric 3’-overhangs by copying a template within the integral RNA subunit of the enzyme. The catalytic core of telomerase consists of the RNA subunit, designated TER, and a catalytic protein subunit designated Telomerase Reverse Transcriptase (TERT). The holoenzyme also contains additional accessory proteins, which are essential for its assembly and enzymatic activity in vivo.

During the synthesis of each telomeric repeat by telomerase, the RNA template is copied till the 5' boundary of the template region is reached. Then, the 3'-end of the newly synthesized DNA dissociates from the template and is repositioned at the beginning of the template for the next round of telomeric repeat synthesis. Telomerase has the unique ability to add multiple copies of the template onto telomeric DNA in a processive manner. To account for the processivity of the enzyme, it has been postulated that the enzyme contains an anchor site that binds upstream sequences of growing DNA chains when their 3'-ends dissociate from the template. In vitro, telomere synthesis by telomerase is mimicked by elongation of single-stranded oligodeoxynucleotide primers.

I used site-specific photocross-linking to map the binding sites for DNA primers in TER within active Tetrahymena telomerase holoenzyme complexes. The mapping was performed at single-nucleotide resolution by a novel technique based on RNase H digestion of RNA-DNA hybrids made with overlapping complementary oligodeoxynucleotides. The mapping data allowed tracing of the DNA path through the telomerase complexes from the template to the TBE region of TER. TBE is known to bind TERT and to be involved in the template 5’ boundary definition. Based on these findings, I propose that upstream sequences of each growing telomeric DNA chain are involved in regulation of its growth arrest at the 5’-end of the RNA template. In addition, the upstream DNA-TBE interaction may serve as an anchor for the subsequent realignment of the 3’-end of the DNA with the 3’-end of the template during the processive elongation of the DNA by the enzyme.