Coordinated DNA dynamics during the human telomerase catalytic cycle.

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• Telomeres protect chromosome ends and shorten with each cell division.
• Telomerase, a reverse transcriptase with an RNA template (hTR), maintains telomere length.
• Telomerase activity is crucial for cellular immortality and is often dysregulated in cancer.

Purpose of the Study:

• To elucidate the mechanism of DNA translocation during telomerase repeat addition processivity (RAP).
• To characterize the kinetic substeps involved in telomere repeat synthesis.
• To understand how the DNA product is repositioned for subsequent rounds of extension.

Main Methods:

• Single-molecule Förster Resonance Energy Transfer (smFRET) assays.
• Nuclease protection assays.
• Monitoring telomere DNA structure and dynamics during the telomerase catalytic cycle.

Main Results:

• Identified a previously uncharacterized kinetic substep in DNA translocation during RAP.
• The 3'-end of the DNA substrate base pairs downstream within the hTR template during this substep.
• DNA primer realignment is not the rate-limiting step for RAP.

Conclusions:

• Telomere repeat synthesis involves a slow conformational change that repositions the RNA:DNA hybrid.
• This repositioning drives the extrusion of the 5'-end of the DNA primer.
• The findings provide new insights into the dynamic mechanism of telomerase function.