title: The N-terminus of hTERT contains a DNA-binding domain and is required for telomerase activity and cellular immortalization.
abstract: Telomerase defers the onset of telomere damage-induced signaling and cellular senescence by adding DNA onto chromosome ends. The ability of telomerase to elongate single-stranded telomeric DNA depends on the reverse transcriptase domain of TERT, and also relies on protein:DNA contacts outside the active site. We purified the N-terminus of human TERT (hTEN) from Escherichia coli, and found that it binds DNA with a preference for telomeric sequence of a certain length and register. hTEN interacted with the C-terminus of hTERT in trans to reconstitute enzymatic activity in vitro. Mutational analysis of hTEN revealed that amino acids Y18 and Q169 were required for telomerase activity in vitro, but not for the interaction with telomere DNA or the C-terminus. These mutants did not reconstitute telomerase activity in cells, maintain telomere length, or extend cellular lifespan. In addition, we found that T116/T117/S118, while dispensable in vitro, were required for cellular immortalization. Thus, the interactions of hTEN with telomere DNA and the C-terminus of hTERT are functionally separable from the role of hTEN in telomere elongation activity in vitro and in vivo, suggesting other roles for the protein and nucleic acid interactions of hTEN within, and possibly outside, the telomerase catalytic core.
authors.affiliation: Department of Medical Biophysics, University of Toronto, Campbell Family Institute for Breast Cancer Research, Ontario Cancer Institute, University Health Network, Toronto, Ontario, M5G 2C1, Canada.
authors.forename: David C F
authors.initials: DC
authors.lastname: Sealey
authors.forename: Le
authors.initials: L
authors.lastname: Zheng
authors.forename: Michael A S
authors.initials: MA
authors.lastname: Taboski
authors.forename: Jennifer
authors.initials: J
authors.lastname: Cruickshank
authors.forename: Mitsuhiko
authors.initials: M
authors.lastname: Ikura
authors.forename: Lea A
authors.initials: LA
authors.lastname: Harrington
mesh_terms: D002460:Cell Line; D002461:Cell Line, Transformed; D049109:Cell Proliferation; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D009154:Mutation; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D019098:Telomerase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016615:Telomere / Q000378:metabolism
chemical_list: D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D004247:DNA; C509186:TERT protein, human; D019098:Telomerase