טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentRomi Erez
SubjectPhysical Mapping of the Binding Site to DNA Primers in the
Protein Sub-unit of Telomerase
DepartmentDepartment of Biology
Supervisor Professor Haim Manor


Abstract

Telomerase is a specialized reverse transcriptase which elongates the single stranded DNA overhangs at the ends of eukaryotic chromosomes. The telomerase core consists of two subunits: an RNA molecule that includes a short sequence, used by the enzyme as a template for DNA synthesis; and a catalytic protein subunit, designated Telomerase Reverse Transcriptase (TERT). The aim of my research was to map the binding site for the DNA substrate in TERT. The mapping was carried out as follows: a 6 mer oligodeoxynucleotide primer, containing a photoreactive nucleotide at position -5, was cross-linked to reconstituted telomerase from Tetrahymena thermophila by UV irradiation. The primer was then elongated with the telomerase by a single 32P-labeled dGMP. Subsequently, the cross-linked TERT was digested with cyanogen bromide (CNBr), which cleaved the protein at 18 specific sites. The digestion conditions were selected such that each molecule was cleaved either once (“single hit” cleavage) or in all 18 sites (complete cleavage). The fragments containing cross-linked radioactive primers were identified by electrophoresis of the cleavage products, followed  by a phosphorimager scan for the presence of radioactive bands. These data revealed that the cross-linked DNA primer is located between amino acids 14-194 in the protein sequence. This region lies outside the reverse transcriptase domain of the enzyme and contains a motif conserved among all telomerases. The newly identified DNA binding site presumably participates in the positioning and stabilization of the DNA molecule along the RNA template during the initial alignment of the DNA in the telomerase-DNA complex.