Telomere DNA G-quadruplex folding within actively extending human telomerase

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• Telomerase maintains telomere length by synthesizing G-rich DNA repeats.
• G-rich sequences can form G-quadruplex (GQ) structures, posing challenges for DNA replication.
• The interplay between telomerase and GQ structures is not fully understood.

Purpose of the Study:

• To investigate how G-quadruplex (GQ) folding of nascent DNA affects telomerase catalysis.
• To elucidate the role of GQ structures in telomere repeat addition and enzyme dissociation.
• To characterize the influence of GQ folding on telomerase function in the presence of POT1-TPP1.

Main Methods:

• Ensemble and single-molecule telomerase assays.
• Experiments using varying DNA primer sequences and potassium (K+) vs. lithium (Li+) salts.
• Single-molecule Förster resonance energy transfer (smFRET) to observe DNA dynamics.
• Kinetic modeling of telomerase time-series data.

Main Results:

• GQ folding of nascent DNA product modulates telomerase catalysis and dissociation rates.
• Changes in DNA product profiles indicate GQ structure formation within the telomerase-DNA complex.
• POT1-TPP1 alters DNA product profiles but does not fully restore activity with Li+.
• smFRET reveals complex DNA structural dynamics during catalysis, dependent on K+ presence.

Conclusions:

• Intrinsic G-quadruplex folding properties of telomere DNA significantly influence telomerase polymerase function.
• GQ folding acts synergistically with POT1-TPP1 to support telomerase activity.
• This study provides a detailed characterization of G-quadruplex modulation of telomerase.