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. 2007;35(4):1155-68.
doi: 10.1093/nar/gkm002. Epub 2007 Jan 30.

The Active Site Residue Valine 867 in Human Telomerase Reverse Transcriptase Influences Nucleotide Incorporation and Fidelity

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Free PMC article

The Active Site Residue Valine 867 in Human Telomerase Reverse Transcriptase Influences Nucleotide Incorporation and Fidelity

William C Drosopoulos et al. Nucleic Acids Res. .
Free PMC article

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.

Figures

Figure 1.
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.
Figure 2.
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.
Figure 3.
Figure 3.
Competitive primer challenge assay with hTERT C-motif mutants. Primer extension reactions were carried out under competitor challenge conditions as detailed in Materials and Methods section. Following 5-min binding of radiolabeled substrate primer, extension reactions were initiated and chased with excess cold competitor primer. 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. Marker sizes (in nucleotides) are indicated. 85 nt R + 3 product band of V867M telomerase is indicated by an arrowhead.
Figure 4.
Figure 4.
Competitive primer challenge assay with hTERT C-motif mutants under reduced nucleotide concentration. Primer extension reactions were carried out under competitor challenge conditions as detailed in Materials and Methods section, except that extension reactions were performed at a dNTP concentration of 50 µM rather than the standard 1 mM. Following 5-min binding of radiolabeled substrate primer, extension reactions were initiated and chased with excess cold competitor primer. 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. Marker sizes (in nucleotides) are indicated.
Figure 5.
Figure 5.
Effect of dTTP concentration on primer extension activity. Primer extension synthesis by wild type and V867M telomerases was measured in primer extension reactions in the presence of increasing concentrations of TTP. Extension reactions containing 1 mM dATP, 1 mM dGTP and TTP at the concentration indicated, were performed under standard conditions as detailed in Materials and Methods section. Representative repeat, R, as well as R + 3 and R + 6 products are indicated, along with potential product alignments with template RNA at right. Nucleotides added during each round of primer elongation are shown in lower case. hTR nucleotide positions are shown next to template sequence. Both wild type (left) and V867M (right) panels are from the same gel.
Figure 6.
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.
Figure 7.
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.
Figure 8.
Figure 8.
Schematic representation of hTERT residue 867 substitutions. Potential orientations of the side chains of the Val (wild type), Met, Thr, Ala 867 residues within the active site of hTERT are depicted. These orientations were generated by mutating the analogous HIV-1 RT residue Met184 in the HIV-1 RT ternary complex structure using the published coordinates (PDB1RTD) of Huang et al. (20). Individual substitutions (M184V, A, or T) were created in the published HIV-1 RT structure using the DeepView/Swiss-pdbViewer program. The ‘best’ fit rotamers are presented.

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