Two-step mechanism involving active-site conformational changes regulates human telomerase DNA binding

Area of Science:

• Biochemistry
• Molecular Biology
• Genetics

Background:

• Telomerase is a ribonucleoprotein enzyme essential for maintaining telomeres and cellular immortality.
• Dyskeratosis congenita, linked to telomerase mutations, highlights telomere dysfunction.
• Understanding telomerase mechanism is vital for cancer research and therapeutic development.

Purpose of the Study:

• To investigate the kinetic and thermodynamic properties of wild-type telomerase and two disease-associated mutations.
• To elucidate the mechanism of telomerase binding to telomeric DNA.
• To validate a homology model of the human telomerase reverse transcriptase domain.

Main Methods:

• Detailed kinetic and thermodynamic analyses of wild-type and mutant telomerase.
• Assessment of primer dissociation rates and DNA affinities.
• Generation and validation of a human telomerase reverse transcriptase domain homology model.

Main Results:

• Telomerase employs a novel two-step mechanism for initial DNA binding involving enzyme conformational changes.
• Mutations P704S and R865H differentially impair telomerase DNA binding.
• P704S affects protein conformational changes, while R865H impacts binding to the DNA 3' region.
• Structural model corroborates the mechanistic defects observed for the mutations.

Conclusions:

• Protein interactions with the telomeric DNA 3' end are critical for telomerase function.
• Enzyme conformational changes play a key role in telomerase DNA binding.
• Naturally occurring disease mutations provide valuable mechanistic insights into telomerase function.