טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentYonit Haviv
SubjectStudies on the Mechanism of Telomeric DNA Extension by
Telomerase
DepartmentDepartment of Biology
Supervisor Full Professor Manor Haim


Abstract

Telomerase is a reverse transcriptase that catalyzes elongation of single-stranded 3’-extensions of telomeres and breakpoints in chromosomes. The catalytic core of telomerase consists of an RNA subunit (TER) including a short template sequence that encodes the single-stranded telomeric repeats and a protein subunit (TERT) containing reverse transcriptase motifs. The enzyme also contains auxiliary proteins.

I have carried out an interference footprinting analysis of interactions of Tetrahymena telomerase reconstituted in vitro from TER and TERT with single-stranded DNA primers within telomerase elongation complexes. In these complexes, the 3’-termini of the primers aligned at 3 different positions along the template: the beginning (3`-terminus), middle and the end (5`-terminus) of the template. The interference footprinting data indicated that in elongation complexes generated with primers aligned at these three positions, the enzyme interacted predominantly with the six or seven 3’-terminal residues in the primers. Similar interactions were also observed by footprinting of primers that were extended by two or three nucleotides. I also found that, at all three positions, these interactions occurred not only with the RNA template region, but also with other regions in the enzyme ribonucleoprotein complex. These interactions reflected the main interface between the primers and the two components of the core enzyme. My data indicated that this interface is shifted downstream relative to the template, along with the active site of the enzyme and the 3’- terminus in the primers.

The observation that in all primers major interactions occurred only with the 3’-terminal residues prompted us to determine the minimal lengths required for primers to be extended by the telomerase. These studies, which are also reported in my thesis, revealed that the minimal lengths were 4, 5 and 6 nucleotides for primers aligned in the beginning, middle and end of the template. These data are compatible with the footprinting data in that they confirmed that interactions with residues upstream of the 6th nucleotide in longer primers are not required for the extension of such primers.

Based on these data and on recent data of others, I present in this work an updated model for primer extension by telomerase.