title: Coordinated DNA dynamics during the human telomerase catalytic cycle.
abstract: The human telomerase reverse transcriptase (hTERT) utilizes a template within the integral RNA subunit (hTR) to direct extension of telomeres. Telomerase exhibits repeat addition processivity (RAP) and must therefore translocate the nascent DNA product into a new RNA:DNA hybrid register to prime each round of telomere repeat synthesis. Here, we use single-molecule FRET and nuclease protection assays to monitor telomere DNA structure and dynamics during the telomerase catalytic cycle. DNA translocation during RAP proceeds through a previously uncharacterized kinetic substep during which the 3'-end of the DNA substrate base pairs downstream within the hTR template. The rate constant for DNA primer realignment reveals this step is not rate limiting for RAP, suggesting a second slow conformational change repositions the RNA:DNA hybrid into the telomerase active site and drives the extrusion of the 5'-end of the DNA primer out of the enzyme complex.
authors.affiliation: Department of Chemistry and Biochemistry, University of California, Santa Cruz, California 95064, USA.
authors.forename: Joseph W
authors.initials: JW
authors.lastname: Parks
authors.affiliation: 1] Department of Chemistry and Biochemistry, University of California, Santa Cruz, California 95064, USA [2] Center for Molecular Biology of RNA, University of California, 1156 High Street, Santa Cruz, California 95064, USA.
authors.forename: Michael D
authors.initials: MD
authors.lastname: Stone
mesh_terms: D055162:Biocatalysis; D020134:Catalytic Domain; D004247:DNA / Q000235:genetics* / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics / Q000378:metabolism; D006801:Humans; D012313:RNA / Q000235:genetics / Q000378:metabolism; D019098:Telomerase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016615:Telomere / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic
chemical_list: D017931:DNA Primers; C422952:telomerase RNA; D012313:RNA; D004247:DNA; C509186:TERT protein, human; D019098:Telomerase