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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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Sequence alignment of TERT protein C motifs and hTERT C-motif mutants. (A) Alignment of TERT protein C-motif sequences from human (36,37), dog (38), mouse (39), rat (GenBank accession no. AAF62177.1), hamster (40), S. pombe (36), E. cuniculi (41), chicken (42), frog (43), T. Thermophila (44,45), E. aediculatus (4), O. trifallax (44), M. crassus (46), C. albicans (EMBL accession no. CAC37831.1) and S. cerevisiae (4). The HIV-1 RT C-motif sequence (47) is also shown. (B) Sequences of hTERT C-motif mutants studied in this work.
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Figure 1: Sequence alignment of TERT protein C motifs and hTERT C-motif mutants. (A) Alignment of TERT protein C-motif sequences from human (36,37), dog (38), mouse (39), rat (GenBank accession no. AAF62177.1), hamster (40), S. pombe (36), E. cuniculi (41), chicken (42), frog (43), T. Thermophila (44,45), E. aediculatus (4), O. trifallax (44), M. crassus (46), C. albicans (EMBL accession no. CAC37831.1) and S. cerevisiae (4). The HIV-1 RT C-motif sequence (47) is also shown. (B) Sequences of hTERT C-motif mutants studied in this work.

Mentions: The initial identification of the TERT protein component of telomerase was facilitated by its homology to other reverse transcriptases (3,4) including human immunodeficiency virus-1 RT (HIV-1 RT). Sequence analysis of TERT proteins reveals the presence of several of the conserved motifs important for RT function. Among these motifs the RT C motif, which contains a common signature sequence ‘YXDD’ (5–7) shared by all retroviral reverse transcriptases, has been shown to be one of the most essential for enzyme activity (8,9). The invariant aspartates in this motif constitute part of an absolutely conserved aspartate triad thought to directly participate in nucleotide addition, coordinated by divalent cations via a two metal ion mechanism (10). TERT proteins also contain a triad of invariant aspartates, two of which are within a ‘hhDD’ (where h = hydrophobic residue) sequence in their C motifs analogous to the YXDD sequence (Figure 1A), that when substituted with alanine result in completely inactive enzymes (4,11,12). The fact that these acidic residues are indispensable underscores their functional importance for telomerase enzymatic activity and suggests that telomerase may also utilize a two metal ion mechanism to catalyze polymerization.Figure 1.


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

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

Sequence alignment of TERT protein C motifs and hTERT C-motif mutants. (A) Alignment of TERT protein C-motif sequences from human (36,37), dog (38), mouse (39), rat (GenBank accession no. AAF62177.1), hamster (40), S. pombe (36), E. cuniculi (41), chicken (42), frog (43), T. Thermophila (44,45), E. aediculatus (4), O. trifallax (44), M. crassus (46), C. albicans (EMBL accession no. CAC37831.1) and S. cerevisiae (4). The HIV-1 RT C-motif sequence (47) is also shown. (B) Sequences of hTERT C-motif mutants studied in this work.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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Figure 1: Sequence alignment of TERT protein C motifs and hTERT C-motif mutants. (A) Alignment of TERT protein C-motif sequences from human (36,37), dog (38), mouse (39), rat (GenBank accession no. AAF62177.1), hamster (40), S. pombe (36), E. cuniculi (41), chicken (42), frog (43), T. Thermophila (44,45), E. aediculatus (4), O. trifallax (44), M. crassus (46), C. albicans (EMBL accession no. CAC37831.1) and S. cerevisiae (4). The HIV-1 RT C-motif sequence (47) is also shown. (B) Sequences of hTERT C-motif mutants studied in this work.
Mentions: The initial identification of the TERT protein component of telomerase was facilitated by its homology to other reverse transcriptases (3,4) including human immunodeficiency virus-1 RT (HIV-1 RT). Sequence analysis of TERT proteins reveals the presence of several of the conserved motifs important for RT function. Among these motifs the RT C motif, which contains a common signature sequence ‘YXDD’ (5–7) shared by all retroviral reverse transcriptases, has been shown to be one of the most essential for enzyme activity (8,9). The invariant aspartates in this motif constitute part of an absolutely conserved aspartate triad thought to directly participate in nucleotide addition, coordinated by divalent cations via a two metal ion mechanism (10). TERT proteins also contain a triad of invariant aspartates, two of which are within a ‘hhDD’ (where h = hydrophobic residue) sequence in their C motifs analogous to the YXDD sequence (Figure 1A), that when substituted with alanine result in completely inactive enzymes (4,11,12). The fact that these acidic residues are indispensable underscores their functional importance for telomerase enzymatic activity and suggests that telomerase may also utilize a two metal ion mechanism to catalyze polymerization.Figure 1.

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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