title: Native gel electrophoresis of human telomerase distinguishes active complexes with or without dyskerin.
abstract: Telomeres, the ends of linear chromosomes, safeguard against genome instability. The enzyme responsible for extension of the telomere 3' terminus is the ribonucleoprotein telomerase. Whereas telomerase activity can be reconstituted in vitro with only the telomerase RNA (hTR) and telomerase reverse transcriptase (TERT), additional components are required in vivo for enzyme assembly, stability and telomere extension activity. One such associated protein, dyskerin, promotes hTR stability in vivo and is the only component to co-purify with active, endogenous human telomerase. We used oligonucleotide-based affinity purification of hTR followed by native gel electrophoresis and in-gel telomerase activity detection to query the composition of telomerase at different purification stringencies. At low salt concentrations (0.1 M NaCl), affinity-purified telomerase was 'supershifted' with an anti-dyskerin antibody, however the association with dyskerin was lost after purification at 0.6 M NaCl, despite the retention of telomerase activity and a comparable yield of hTR. The interaction of purified hTR and dyskerin in vitro displayed a similar salt-sensitive interaction. These results demonstrate that endogenous human telomerase, once assembled and active, does not require dyskerin for catalytic activity. Native gel electrophoresis may prove useful in the characterization of telomerase complexes under various physiological conditions.
authors.affiliation: Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh EH9 3JR, UK.
authors.forename: Laura
authors.initials: L
authors.lastname: Gardano
authors.forename: Linda
authors.initials: L
authors.lastname: Holland
authors.forename: Rena
authors.initials: R
authors.lastname: Oulton
authors.forename: Thierry
authors.initials: T
authors.lastname: Le Bihan
authors.forename: Lea
authors.initials: L
authors.lastname: Harrington
mesh_terms: D000906:Antibodies; D018797:Cell Cycle Proteins / Q000276:immunology / Q000378:metabolism*; D045744:Cell Line, Tumor; D004586:Electrophoresis; D006801:Humans; D009687:Nuclear Proteins / Q000276:immunology / Q000378:metabolism*; D009841:Oligonucleotides; D012965:Sodium Chloride / Q000737:chemistry; D019098:Telomerase / Q000032:analysis / Q000302:isolation & purification* / Q000378:metabolism*
chemical_list: D000906:Antibodies; D018797:Cell Cycle Proteins; C112148:DKC1 protein, human; D009687:Nuclear Proteins; D009841:Oligonucleotides; D012965:Sodium Chloride; D019098:Telomerase