Mechanisms of the Human Telomerase Catalytic Cycle
- Author(s): Wu, Robert Alexander
- Advisor(s): Collins, Kathleen
- et al.
At the ends of every linear chromosome, genomic integrity is threatened by incomplete
DNA synthesis by the replisome and the potential for inappropriate DNA break repair.
Eukaryotic cells control these reactions through the function of telomeres. Maintenance
of the characteristic DNA repeat tracts that form telomeres requires the specialized
reverse transcriptase telomerase, with its active site in the protein subunit TERT and the
template for DNA synthesis in the integral RNA subunit. Many telomerases can extend a
chromosome 3' end by processive addition of single-stranded repeats. This processive
telomeric repeat synthesis requires a specialized telomerase catalytic cycle, involving
nucleic acid handling specificities not found in any other polymerase. Developing new
approaches of subunit fluorescence labeling for single-molecule analysis, I show that
human telomerase reconstitution generates mixtures of complexes of varying TERT
subunit stoichiometry but activity requires a complex containing only one TERT
molecule. This establishes conservation of active telomerase subunit architecture across
phylogeny. Using TERT and RNA domain-complementation assays to sensitize for
primer-template duplex use by the telomerase active site and a direct footprinting assay
for telomerase association with product DNA, I uncover mechanisms by which TERT
domains and RNA motifs interact to specify telomeric repeat synthesis. This work
develops a new model for specialized primer-template duplex sensing during the human
telomerase catalytic cycle.