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Mechanisms of the Human Telomerase Catalytic Cycle


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.

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