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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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Effect of hTR template sequence on primer extension by 867M hTERT. (A) Wild type and V867M hTERT proteins were reconstituted with wild type and template mutant (MH) hTR RNAs (23) shown. (B) Primer extension reactions were carried out under competitor challenge conditions as detailed in Materials and Methods section. Post-chase aliquots were taken at 3 and 30 min and analyzed via PAGE. Pre-chased lane: 30-min extension reaction where excess competitor primer was added before adding IVR wild-type telomerase. Lysate lane: extension reaction with control IVR containing RRL only. (C) Enlarged view of region between 48 and 75 nt. Sequence of the WT hTR template (nucleotides 46–51) is shown along with the sequence of the wild-type product. The number of nucleotides added onto a previously copied full repeat is indicated in parentheses. Arrowheads indicate product accumulation prior to copying a template A residue.
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Figure 6: Effect of hTR template sequence on primer extension by 867M hTERT. (A) Wild type and V867M hTERT proteins were reconstituted with wild type and template mutant (MH) hTR RNAs (23) shown. (B) Primer extension reactions were carried out under competitor challenge conditions as detailed in Materials and Methods section. Post-chase aliquots were taken at 3 and 30 min and analyzed via PAGE. Pre-chased lane: 30-min extension reaction where excess competitor primer was added before adding IVR wild-type telomerase. Lysate lane: extension reaction with control IVR containing RRL only. (C) Enlarged view of region between 48 and 75 nt. Sequence of the WT hTR template (nucleotides 46–51) is shown along with the sequence of the wild-type product. The number of nucleotides added onto a previously copied full repeat is indicated in parentheses. Arrowheads indicate product accumulation prior to copying a template A residue.

Mentions: Based on the activity assays it appeared that the reduction in repeat extension rate was correlated to the difficulty Val867 mutants had copying past residue A48. This raised the question of whether the copying defect is specific to residue A48 (or hTR nucleotide 48) or a general defect copying template A residues. In order to answer this question, template copying by V867M hTERT was examined using a panel of hTR mutants with modified template sequences described previously (23). The template sequences of these mutant hTRs contained different substitutions within the template sequence 49AAU47, resulting in sequences with zero to three template A residues (Figure 6A). Four out of the five mutants that were tested contained A48U substitutions. In addition, as these hTR template mutants were designed to allow for efficient processive repeat synthesis, the alignment sequences (nucleotides 55AAU53) in these mutants were also modified to reflect the changes to the templating nucleotides (49AAU47) (Figure 6A).Figure 6.


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

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

Effect of hTR template sequence on primer extension by 867M hTERT. (A) Wild type and V867M hTERT proteins were reconstituted with wild type and template mutant (MH) hTR RNAs (23) shown. (B) Primer extension reactions were carried out under competitor challenge conditions as detailed in Materials and Methods section. Post-chase aliquots were taken at 3 and 30 min and analyzed via PAGE. Pre-chased lane: 30-min extension reaction where excess competitor primer was added before adding IVR wild-type telomerase. Lysate lane: extension reaction with control IVR containing RRL only. (C) Enlarged view of region between 48 and 75 nt. Sequence of the WT hTR template (nucleotides 46–51) is shown along with the sequence of the wild-type product. The number of nucleotides added onto a previously copied full repeat is indicated in parentheses. Arrowheads indicate product accumulation prior to copying a template A residue.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

Figure 6: Effect of hTR template sequence on primer extension by 867M hTERT. (A) Wild type and V867M hTERT proteins were reconstituted with wild type and template mutant (MH) hTR RNAs (23) shown. (B) Primer extension reactions were carried out under competitor challenge conditions as detailed in Materials and Methods section. Post-chase aliquots were taken at 3 and 30 min and analyzed via PAGE. Pre-chased lane: 30-min extension reaction where excess competitor primer was added before adding IVR wild-type telomerase. Lysate lane: extension reaction with control IVR containing RRL only. (C) Enlarged view of region between 48 and 75 nt. Sequence of the WT hTR template (nucleotides 46–51) is shown along with the sequence of the wild-type product. The number of nucleotides added onto a previously copied full repeat is indicated in parentheses. Arrowheads indicate product accumulation prior to copying a template A residue.
Mentions: Based on the activity assays it appeared that the reduction in repeat extension rate was correlated to the difficulty Val867 mutants had copying past residue A48. This raised the question of whether the copying defect is specific to residue A48 (or hTR nucleotide 48) or a general defect copying template A residues. In order to answer this question, template copying by V867M hTERT was examined using a panel of hTR mutants with modified template sequences described previously (23). The template sequences of these mutant hTRs contained different substitutions within the template sequence 49AAU47, resulting in sequences with zero to three template A residues (Figure 6A). Four out of the five mutants that were tested contained A48U substitutions. In addition, as these hTR template mutants were designed to allow for efficient processive repeat synthesis, the alignment sequences (nucleotides 55AAU53) in these mutants were also modified to reflect the changes to the templating nucleotides (49AAU47) (Figure 6A).Figure 6.

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