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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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Primer extension by hTERT C-motif mutants. (A) In vitro reconstituted telomerase mutants were assayed for telomerase activity via direct primer extension as described in Materials and Methods section. Lysate lane: extension reaction with control IVR containing RRL only. Unextended d(TTAGGG)3 substrate primer (P) within each reaction served as loading control (as it represented >95% of the recovered material). Numbers on left (+4, +10, etc.) indicate the positions of products corresponding to the end of each round of template copying (expressed as number of nucleotides added to the primer). Marker sizes (in nucleotides) are indicated. A representative repeat + 3 (R + 3) product is indicated by arrowhead. (B) Schematic diagram of major synthesis products from primer extension reactions. Potential product alignments of R + 3 and repeat + 6 (R + 6) products with the template RNA are shown. Nucleotides added during each round of primer elongation are shown in lower case. hTR nucleotide positions are indicated next to template sequence.
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Figure 2: Primer extension by hTERT C-motif mutants. (A) In vitro reconstituted telomerase mutants were assayed for telomerase activity via direct primer extension as described in Materials and Methods section. Lysate lane: extension reaction with control IVR containing RRL only. Unextended d(TTAGGG)3 substrate primer (P) within each reaction served as loading control (as it represented >95% of the recovered material). Numbers on left (+4, +10, etc.) indicate the positions of products corresponding to the end of each round of template copying (expressed as number of nucleotides added to the primer). Marker sizes (in nucleotides) are indicated. A representative repeat + 3 (R + 3) product is indicated by arrowhead. (B) Schematic diagram of major synthesis products from primer extension reactions. Potential product alignments of R + 3 and repeat + 6 (R + 6) products with the template RNA are shown. Nucleotides added during each round of primer elongation are shown in lower case. hTR nucleotide positions are indicated next to template sequence.

Mentions: To assess the contribution of the hTERT C-motif residues Leu866 and Val867 to telomerase enzymatic activity and function, a panel of hTERT protein mutants was generated (Figure 1B). These mutants included L866Y, V867A, M, or T, and, the double substitution mutants L866Y/V867M and L866Y/V867T. Among these variants were mutants with substitutions that resulted in hTERT proteins whose C-motif sequences were converted to ciliate (LTDD) and yeast (LADD) TERT sequences, as well as to the HIV-1 RT C-motif (YMDD) sequence. Reconstituted telomerase complexes were assembled in rabbit reticulocyte lysates from hTERT protein synthesized in vitro in the presence of purified, in vitro transcribed human TR (hTR) RNA. The reconstituted enzymes were then assayed for telomerase activity in a direct primer extension assay using radiolabeled telomeric (d(TTAGGG)3) substrate primer. All of the mutants were active to some extent (Figure 2, Table 1), although in every case overall activity was lower than with wild-type hTERT. While some substitutions resulted in modest reductions in activity (L866Y) others had severe effects (V867T) (Table 1). Interestingly, the double substitutions L866Y/V867M and L866Y/V867T yielded enzymes with activities at least as active as the single 867M and 867T substitutions.Figure 2.


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

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

Primer extension by hTERT C-motif mutants. (A) In vitro reconstituted telomerase mutants were assayed for telomerase activity via direct primer extension as described in Materials and Methods section. Lysate lane: extension reaction with control IVR containing RRL only. Unextended d(TTAGGG)3 substrate primer (P) within each reaction served as loading control (as it represented >95% of the recovered material). Numbers on left (+4, +10, etc.) indicate the positions of products corresponding to the end of each round of template copying (expressed as number of nucleotides added to the primer). Marker sizes (in nucleotides) are indicated. A representative repeat + 3 (R + 3) product is indicated by arrowhead. (B) Schematic diagram of major synthesis products from primer extension reactions. Potential product alignments of R + 3 and repeat + 6 (R + 6) products with the template RNA are shown. Nucleotides added during each round of primer elongation are shown in lower case. hTR nucleotide positions are indicated next to template sequence.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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Figure 2: Primer extension by hTERT C-motif mutants. (A) In vitro reconstituted telomerase mutants were assayed for telomerase activity via direct primer extension as described in Materials and Methods section. Lysate lane: extension reaction with control IVR containing RRL only. Unextended d(TTAGGG)3 substrate primer (P) within each reaction served as loading control (as it represented >95% of the recovered material). Numbers on left (+4, +10, etc.) indicate the positions of products corresponding to the end of each round of template copying (expressed as number of nucleotides added to the primer). Marker sizes (in nucleotides) are indicated. A representative repeat + 3 (R + 3) product is indicated by arrowhead. (B) Schematic diagram of major synthesis products from primer extension reactions. Potential product alignments of R + 3 and repeat + 6 (R + 6) products with the template RNA are shown. Nucleotides added during each round of primer elongation are shown in lower case. hTR nucleotide positions are indicated next to template sequence.
Mentions: To assess the contribution of the hTERT C-motif residues Leu866 and Val867 to telomerase enzymatic activity and function, a panel of hTERT protein mutants was generated (Figure 1B). These mutants included L866Y, V867A, M, or T, and, the double substitution mutants L866Y/V867M and L866Y/V867T. Among these variants were mutants with substitutions that resulted in hTERT proteins whose C-motif sequences were converted to ciliate (LTDD) and yeast (LADD) TERT sequences, as well as to the HIV-1 RT C-motif (YMDD) sequence. Reconstituted telomerase complexes were assembled in rabbit reticulocyte lysates from hTERT protein synthesized in vitro in the presence of purified, in vitro transcribed human TR (hTR) RNA. The reconstituted enzymes were then assayed for telomerase activity in a direct primer extension assay using radiolabeled telomeric (d(TTAGGG)3) substrate primer. All of the mutants were active to some extent (Figure 2, Table 1), although in every case overall activity was lower than with wild-type hTERT. While some substitutions resulted in modest reductions in activity (L866Y) others had severe effects (V867T) (Table 1). Interestingly, the double substitutions L866Y/V867M and L866Y/V867T yielded enzymes with activities at least as active as the single 867M and 867T substitutions.Figure 2.

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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