A single nucleotide incorporation step limits human telomerase repeat addition activity

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• Human telomerase synthesizes telomeric DNA repeats (GGTTAG)n onto chromosome ends using telomerase RNA (hTR).
• Telomerase exhibits significantly slower processive DNA synthesis compared to other DNA polymerases.

Purpose of the Study:

• To investigate the mechanism behind telomerase's slow DNA synthesis rate.
• To identify factors influencing telomerase processivity and catalytic efficiency.
• To explore therapeutic strategies for enhancing telomerase activity.

Main Methods:

• Characterization of telomerase kinetics and substrate specificity.
• Site-directed mutagenesis of the telomerase RNA template (hTR-51U).
• Assays measuring telomerase repeat addition activity and processivity under varying nucleotide concentrations.

Main Results:

• A template-embedded pause signal significantly increases the K_M for the first nucleotide incorporation, limiting repeat addition processivity and rate.
• Augmented concentrations of deoxyguanosine triphosphate (dGTP) or deoxyguanosine diphosphate (dGDP) alleviate the slow incorporation step.
• Mutating the template to incorporate adenosine instead of guanosine shifted stimulation to deoxyadenosine triphosphate (dATP)-dependent, confirming the pause signal's role.
• Telomerase lacking the pause signal demonstrated high efficiency and lacked dGTP stimulation.

Conclusions:

• The initial nucleotide incorporation step is a rate-limiting factor in the telomerase catalytic cycle.
• Nucleoside diphosphates can serve as substrates, and dGDP specifically stimulates telomerase activity.
• Targeting the first nucleotide incorporation step offers a potential therapeutic strategy for modulating telomerase activity.