Multiple DNA-binding sites in Tetrahymena telomerase

Area of Science:

• Molecular Biology
• Enzymology
• Genetics

Background:

• Telomerase is a ribonucleoprotein enzyme essential for maintaining chromosome stability.
• Repeat addition processivity in telomerase relies on interactions with DNA substrates at anchor sites.
• Understanding telomerase-TERT DNA interactions is key to its function in chromosome maintenance.

Purpose of the Study:

• To quantify DNA-binding affinities of recombinant Tetrahymena telomerase protein subunit (TERT) regions.
• To investigate the role of multiple anchor sites in telomerase-DNA interactions.
• To elucidate the contribution of different TERT domains to telomeric DNA binding.

Main Methods:

• Development of a direct, quantitative equilibrium primer-binding assay.
• Measurement of DNA-binding affinities for recombinant Tetrahymena telomerase (TERT) domains.
• Analysis of primer length effects on binding affinity (K(m) and K(d)).

Main Results:

• Specific telomeric DNA-binding sites identified in at least four TERT regions (TEN, RBD, RT, C-terminal).
• High-affinity DNA binding observed (K(d) ≈ 8 nM), increasing with primer length.
• Multiple anchor sites demonstrated, with cooperativity between TEN and RNA-binding domains, and low affinity for the TEN domain alone.

Conclusions:

• Tetrahymena telomerase (TERT) possesses multiple specific DNA-binding sites contributing to high-affinity interactions.
• The enzyme utilizes different DNA-binding sites throughout the telomeric DNA addition cycle.
• Findings provide insights into the mechanism of telomerase repeat addition processivity.