DNA‐binding determinants and cellular thresholds for human telomerase repeat addition processivity

• Published in: The EMBO journal 2017-07, Vol.36 (13), p.1908-1927
• Main Authors: Wu, Robert Alexander, Tam, Jane, Collins, Kathleen
• Format: Article
• Language: English
• Subjects: Binding; Biochemistry; Biological analysis; Catalytic Domain; Chemical synthesis; Chromosomes; Chromosomes, Human - metabolism; Deoxyribonucleic acid; Determinants; DNA; DNA–RNA duplex; Elongation; EMBO13; Exonuclease; Humans; Lower bounds; Maintenance; Models, Biological; Models, Molecular; Nucleotides; Repetitive Sequences, Nucleic Acid; reverse transcriptase; Ribonucleic acid; RNA; RNA-directed DNA polymerase; Single-stranded DNA; Telomerase; Telomerase - chemistry; Telomerase - metabolism; telomere maintenance; Telomeres; Thresholds; Translocation
• ISSN: 0261-4189; 1460-2075
• DOI: 10.15252/embj.201796887

The reverse transcriptase telomerase adds telomeric repeats to chromosome ends. Purified human telomerase catalyzes processive repeat synthesis, which could restore the full ~100 nucleotides of (T 2 AG 3 ) n lost from replicated chromosome ends as a single elongation event. Processivity inhibition is proposed to be a basis of human disease, but the impacts of different levels of processivity on telomere maintenance have not been examined. Here, we delineate side chains in the telomerase active‐site cavity important for repeat addition processivity, determine how they contribute to duplex and single‐stranded DNA handling, and test the cellular consequences of partial or complete loss of repeat addition processivity for telomere maintenance. Biochemical findings oblige a new model for DNA and RNA handling dynamics in processive repeat synthesis. Biological analyses implicate repeat addition processivity as essential for telomerase function. However, telomeres can be maintained by telomerases with lower than wild‐type processivity. Furthermore, telomerases with low processivity dramatically elongate telomeres when overexpressed. These studies reveal distinct consequences of changes in telomerase repeat addition processivity and expression level on telomere elongation and length maintenance. Synopsis Repeat addition processivity (RAP) allows telomerase to restore replicated telomeres in a single elongation event. Identification of telomerase DNA binding determinants for RAP allows the study of its importance for telomere maintenance in human cells. Product–template duplex handling by human telomerase changes with each successive dNTP incorporation. Lengths of exonuclease‐protected telomerase products suggest a maximal duplex of about seven base‐pairs. Active site cavity recognition of the product–template duplex formed by the template 5′ end promotes unpairing of the template 3′ end. Template‐paired DNA engages a single‐stranded DNA retention surface that retains product DNA during template translocation for RAP. Within upper and lower bounds, human telomerase RAP determines the set‐point for telomere length maintenance. Graphical Abstract Telomerase mutants with reduced processivity may still maintain telomeres, and at higher expression levels can even cause telomere over‐elongation in human cells.