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

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 aspart...

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Main Authors:	Drosopoulos, William C., Prasad, Vinayaka R.
Format:	Online
Language:	English
Published:	Oxford University Press 2007
Online Access:	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1851649/

id	pubmed-1851649
recordtype	oai_dc
spelling	pubmed-18516492007-04-26 The active site residue Valine 867 in human telomerase reverse transcriptase influences nucleotide incorporation and fidelity Drosopoulos, William C. Prasad, Vinayaka R. Nucleic Acid Enzymes 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. Oxford University Press 2007-02 2007-01-30 /pmc/articles/PMC1851649/ /pubmed/17264120 http://dx.doi.org/10.1093/nar/gkm002 Text en © 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.
repository_type	Open Access Journal
institution_category	Foreign Institution
institution	US National Center for Biotechnology Information
building	NCBI PubMed
collection	Online Access
language	English
format	Online
author	Drosopoulos, William C. Prasad, Vinayaka R.
spellingShingle	Drosopoulos, William C. Prasad, Vinayaka R. The active site residue Valine 867 in human telomerase reverse transcriptase influences nucleotide incorporation and fidelity
author_facet	Drosopoulos, William C. Prasad, Vinayaka R.
author_sort	Drosopoulos, William C.
title	The active site residue Valine 867 in human telomerase reverse transcriptase influences nucleotide incorporation and fidelity
title_short	The active site residue Valine 867 in human telomerase reverse transcriptase influences nucleotide incorporation and fidelity
title_full	The active site residue Valine 867 in human telomerase reverse transcriptase influences nucleotide incorporation and fidelity
title_fullStr	The active site residue Valine 867 in human telomerase reverse transcriptase influences nucleotide incorporation and fidelity
title_full_unstemmed	The active site residue Valine 867 in human telomerase reverse transcriptase influences nucleotide incorporation and fidelity
title_sort	active site residue valine 867 in human telomerase reverse transcriptase influences nucleotide incorporation and fidelity
description	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.
publisher	Oxford University Press
publishDate	2007
url	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1851649/
_version_	1611395619929194496

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

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