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J. Biol. Chem., Vol. 279, Issue 51, 53770-53781, December 17, 2004

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Processive Utilization of the Human Telomerase Template

LACK OF A REQUIREMENT FOR TEMPLATE SWITCHING^*

Melissa A. Rivera and Elizabeth H. Blackburn

From the Department of Biochemistry and Biophysics, University of California San Francisco, California 94143-2200

The ribonucleoprotein telomerase is a specialized reverse transcriptase minimally composed of an RNA, TER, and a protein catalytic subunit, TERT. The TER and TERT subunits of telomerase associate to form a dimeric enzyme in several organisms, including human. A small portion of TER, the template domain, is used by telomerase for the synthesis of tandem repeats of telomeric DNA. We studied some of the requirements for processive template usage by human telomerase. A blunt-ended duplex DNA primer was not utilized by telomerase. With a duplex telomeric DNA primer, a single-stranded 3' overhang with a minimum length of 6 bases was required for efficient priming activity. Large substitutions in the human TER templating domain did not abolish enzymatic activity, although insertion of two residues into this sequence reduced processivity, as did a template mutation that results in a mismatch between the template region used for copying DNA and the region used for alignment of the substrate primer. Finally, by using a complementary pair of catalytically inactive telomerase RNA pseudoknot mutants in combination with a marked template, we demonstrated that processive synthesis by an obligatory dimer of human telomerase does not require template switching. These results indicate that processive template usage by human telomerase, like that of Tetrahymena telomerase, is strongly dependent on the base identities in the template domain and that a dimeric human telomerase can processively utilize a single template.

Received for publication, July 9, 2004 , and in revised form, September 27, 2004.

^* This work was supported by grants from the National Institutes of Health and the Steven and Michelle Kirsch Foundation (to E. H. B.) and by a National Science Foundation predoctoral fellowship (to M. A. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Present address: Dept. of Molecular Genetics and Microbiology, University of Texas, Austin, TX 78712.

To whom correspondence should be addressed. Tel.: 415-476-4912; Fax: 415-514-2913; E-mail: telomer{at}itsa.ucsf.edu.

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