Functional reconstitution of wild-type and mutant Tetrahymena telomerase

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dc.contributor.author Greider, Carol W.
dc.contributor.author Autexier, Chantal
dc.date.accessioned 2009-11-06T21:10:26Z
dc.date.available 2009-11-06T21:10:26Z
dc.date.issued 1994-03-01
dc.identifier.citation Autexier, Chantal and Carol W. Greider. "Functional reconstitution of wild-type and mutant Tetrahymena telomerase" Genes & Development 8 (5) (1994 March 1): 563-575. Originally published: doi: 10.1101/gad.8.5.563 en
dc.identifier.uri http://jhir.library.jhu.edu/handle/1774.2/33549
dc.description.abstract Telomerase is a ribonucleoprotein that catalyzes telomere elongation in vitro and in vivo. The 159-nucleotide RNA component of Tetrahymena telomerase contains the sequence 5'-CAACCCCAA-3' ("template region"), which serves as a template for the addition of the sequence d(TTGGGG)n to Tetrahymena telomeres. To dissect the Tetrahymena telomerase enzyme mechanism, we developed a functional in vitro reconstitution assay. After removal of the essential telomerase RNA by micrococcal nuclease digestion of partially purified telomerase, the addition of in vitro-transcribed telomerase RNA reconstituted telomerase activity. The reconstituted activity was processive and showed the same primer specificities as native telomerase. Mutants in the RNA template region were tested in reconstitution assays to determine the role of the residues in this region in primer recognition and elongation. Two template mutants, encoding the sequences 5'-UAACCCCAA-3' and 5'-UAACCCUAA-3', specified the incorporation of dATP into the sequence d(TTAGGG). Telomerase reconstituted with a template mutant encoding the sequence 5'-CAACCCUAA-3' did not specify dATP incorporation and elongation by this mutant was not terminated by the addition of ddATP. In addition, a template mutant encoding the sequence 5'-CGGCCCCAA-3' specified the incorporation of ddCTP but not ddTTP while a mutant encoding the sequence 5'-CAACCCCGG-3' specified the incorporation of ddTTP but not ddCTP. These data suggest that only the most 5' six residues of the template region dictate the addition of telomeric repeats. en
dc.description.provenance Submitted by David Reynolds (davidr@jhu.edu) on 2009-11-06T21:10:26Z No. of bitstreams: 1 GenesAndDevelop1994.pdf: 2564875 bytes, checksum: ad7ab3d9a068771cfe6e11b18e3f015f (MD5) en
dc.description.provenance Made available in DSpace on 2009-11-06T21:10:26Z (GMT). No. of bitstreams: 1 GenesAndDevelop1994.pdf: 2564875 bytes, checksum: ad7ab3d9a068771cfe6e11b18e3f015f (MD5) Previous issue date: 1994-03-01 en
dc.language.iso en_US en
dc.publisher Cold Spring Harbor Laboratory Press en
dc.subject Tetrahymena/enzymology en
dc.subject DNA Nucleotidylexotransferase /metabolism en
dc.title Functional reconstitution of wild-type and mutant Tetrahymena telomerase en
dc.type Article en

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