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

Drosopoulos WC, Prasad VR - Nucleic Acids Res. (2007)

Bottom Line: All Val867 substitutions examined (Ala, Met, Thr) led to reduced repeat extension rates, ranging from approximately 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA. drosopou@aecom.yu.edu

ABSTRACT
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 approximately 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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Nucleotide exclusion assay with hTERT C-motif mutants. Primer extension reactions were performed under standard conditions with all three substrate nucleotides (dATP, dGTP and TTP) present (+all), only dGTP and TTP present (−dA), or only dATP and TTP present (−dG) in the extension reaction. The sequence of correctly copied product along with the number of nucleotides added to the radiolabeled primer (P*) is indicated. (B) Lower exposure of (A). Expected full-length products of accurate extension synthesis in the absence of dATP (−dA product) and dGTP (−dG product) are shown, with newly copied nucleotides in lower case. The number of nucleotides added to the radiolabeled primer resulting from extension by telomerase is indicated in parentheses.
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Figure 7: Nucleotide exclusion assay with hTERT C-motif mutants. Primer extension reactions were performed under standard conditions with all three substrate nucleotides (dATP, dGTP and TTP) present (+all), only dGTP and TTP present (−dA), or only dATP and TTP present (−dG) in the extension reaction. The sequence of correctly copied product along with the number of nucleotides added to the radiolabeled primer (P*) is indicated. (B) Lower exposure of (A). Expected full-length products of accurate extension synthesis in the absence of dATP (−dA product) and dGTP (−dG product) are shown, with newly copied nucleotides in lower case. The number of nucleotides added to the radiolabeled primer resulting from extension by telomerase is indicated in parentheses.

Mentions: In contrast, reactions lacking dATP or dGTP, revealed major differences in post-barrier site synthesis. With the exception of the L866Y mutant, all of the mutants exhibited significantly lower primer extension past both the ‘U’ barrier site (U47) and the ‘C’ barrier site (C46) compared to wild-type hTERT (Figure 7). In the absence of dATP both wild type and L866Y telomerase could repeatedly extend beyond the U47 barrier position (Figure 7A, WT and L866Y −dA lanes) leading to extension products of over 150 nt (data not shown). Under identical conditions, template copying of U47 by all mutants containing Val867 substitutions was much less efficient, with essentially no extension proceeding beyond the third encounter of the barrier (Figure 7A, −dA lanes). The Val867 mutants were also less efficient than wild type or the L866Y mutant at primer extension past U47 in the absence of dGTP (Figure 7B and C, compare −dG lanes). Careful examination of the post-barrier synthesis products of both dATP and dGTP-excluded reactions (Figure 7A and B) reveals a shift in migration relative to the corresponding products of reactions with all nucleotides present, indicative of true misincorporation.Figure 7.


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

Drosopoulos WC, Prasad VR - Nucleic Acids Res. (2007)

Nucleotide exclusion assay with hTERT C-motif mutants. Primer extension reactions were performed under standard conditions with all three substrate nucleotides (dATP, dGTP and TTP) present (+all), only dGTP and TTP present (−dA), or only dATP and TTP present (−dG) in the extension reaction. The sequence of correctly copied product along with the number of nucleotides added to the radiolabeled primer (P*) is indicated. (B) Lower exposure of (A). Expected full-length products of accurate extension synthesis in the absence of dATP (−dA product) and dGTP (−dG product) are shown, with newly copied nucleotides in lower case. The number of nucleotides added to the radiolabeled primer resulting from extension by telomerase is indicated in parentheses.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1851649&req=5

Figure 7: Nucleotide exclusion assay with hTERT C-motif mutants. Primer extension reactions were performed under standard conditions with all three substrate nucleotides (dATP, dGTP and TTP) present (+all), only dGTP and TTP present (−dA), or only dATP and TTP present (−dG) in the extension reaction. The sequence of correctly copied product along with the number of nucleotides added to the radiolabeled primer (P*) is indicated. (B) Lower exposure of (A). Expected full-length products of accurate extension synthesis in the absence of dATP (−dA product) and dGTP (−dG product) are shown, with newly copied nucleotides in lower case. The number of nucleotides added to the radiolabeled primer resulting from extension by telomerase is indicated in parentheses.
Mentions: In contrast, reactions lacking dATP or dGTP, revealed major differences in post-barrier site synthesis. With the exception of the L866Y mutant, all of the mutants exhibited significantly lower primer extension past both the ‘U’ barrier site (U47) and the ‘C’ barrier site (C46) compared to wild-type hTERT (Figure 7). In the absence of dATP both wild type and L866Y telomerase could repeatedly extend beyond the U47 barrier position (Figure 7A, WT and L866Y −dA lanes) leading to extension products of over 150 nt (data not shown). Under identical conditions, template copying of U47 by all mutants containing Val867 substitutions was much less efficient, with essentially no extension proceeding beyond the third encounter of the barrier (Figure 7A, −dA lanes). The Val867 mutants were also less efficient than wild type or the L866Y mutant at primer extension past U47 in the absence of dGTP (Figure 7B and C, compare −dG lanes). Careful examination of the post-barrier synthesis products of both dATP and dGTP-excluded reactions (Figure 7A and B) reveals a shift in migration relative to the corresponding products of reactions with all nucleotides present, indicative of true misincorporation.Figure 7.

Bottom Line: All Val867 substitutions examined (Ala, Met, Thr) led to reduced repeat extension rates, ranging from approximately 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA. drosopou@aecom.yu.edu

ABSTRACT
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 approximately 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.

Show MeSH
Related in: MedlinePlus