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Nucleic Acids Research Advance Access published online on January 30, 2007
Nucleic Acids Research, doi:10.1093/nar/gkm002
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© 2007 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Nucleic Acid Enzymes

The active site residue Valine 867 in human telomerase reverse transcriptase influences nucleotide incorporation and fidelity

William C. Drosopoulos* and Vinayaka R. Prasad

Department of Microbiology and Immunology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA

*To whom correspondence should be addressed. Tel: +1 718 430 2506; Fax: 1-718-430-8976; E-mail: drosopou{at}aecom.yu.edu Correspondence may also be addressed to Vinayaka R. Prasad. Tel: +1 718 430 2517; Fax: 1-718-430-8976; E-mail: prasad{at}aecom.yu.edu. Correspondence may also be addressed to Vinayaka R. Prasad. +1 718 430 2517; prasad{at}aecom.yu.edu

Received September 6, 2006. Revised December 22, 2006. Accepted December 22, 2006.

Human telomerase reverse transcriptase (hTERT), the catalytic subunit of human telomerase, contains conserved motifs common to retroviral reverse transcriptases and telomerases. Within the C motif of hTERT is the Leu866-Val867-Asp868-Asp869 tetrapeptide that includes a catalytically essential aspartate dyad. Site-directed mutagenesis of Tyr183 and Met184 residues in HIV-1 RT, residues analogous to Leu866 and Val867, revealed that they are key determinants of nucleotide binding, processivity and fidelity. In this study, we show that substitutions at Val867 lead to significant changes in overall enzyme activity and telomere repeat extension rate, but have little effect on polymerase processivity. All Val867 substitutions examined (Ala, Met, Thr) led to reduced repeat extension rates, ranging from ~20 to 50% of the wild-type rate. Reconstitution of V867M hTERT and telomerase RNAs (TRs) with mutated template sequences revealed the effect on extension rate was associated with a template copying defect specific to template A residues. Furthermore, the Val867 hTERT mutants also displayed increased nucleotide incorporation fidelity, implicating Val867 as a determinant of telomerase fidelity. These findings suggest that by evolving to have a valine at position 867, the wild-type hTERT protein may have partially compromised polymerase fidelity for optimal and rapid repeat synthesis.


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