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. 2003 Jul 15;31(14):4059-70.
doi: 10.1093/nar/gkg437.

The C terminus of the human telomerase reverse transcriptase is a determinant of enzyme processivity

Affiliations

The C terminus of the human telomerase reverse transcriptase is a determinant of enzyme processivity

Sylvain Huard et al. Nucleic Acids Res. .

Abstract

The catalytic subunit of telomerase (TERT) contains conserved reverse transcriptase-like motifs but N- and C-terminal regions unique to telomerases. Despite weak sequence conservation, the C terminus of TERTs from various organisms has been implicated in telomerase-specific functions, including telomerase activity, functional multimerization with other TERT molecules, enzyme processivity and telomere length maintenance. We studied hTERT proteins containing small C-terminal deletions or substitutions to identify and characterize hTERT domains mediating telomerase activity, hTERT multimerization and processivity. Using sequence alignment of five vertebrate TERTs and Arabidopsis thaliana TERT, we identified blocks of highly conserved amino acids that were required for human telomerase activity and functional hTERT complementation. We adapted the non-PCR-based telomerase elongation assay to characterize telomerase expressed and reconstituted in the in vitro transcription/translation rabbit reticulocyte lysate system. Using this assay, we found that the hTERT C terminus, like the C terminus of Saccharomyces cerevisiae TERT, contributes to successive nucleotide addition within a single 6-base telomeric repeat (type I processivity). Certain mutations in the hTERT C terminus also reduced the repetitive addition of multiple telomeric repeats (type II processivity). Our results suggest a functionally conserved role for the TERT C terminus in telomerase enzyme processivity.

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Figures

Figure 1
Figure 1
Map of the hTERT C terminus, location of C-terminal mutations, and sequence alignment of the C termini of vertebrate and A.thaliana TERTs. (A) Alignment of the C-terminal amino acid sequences of five vertebrate TERTs and A.thaliana TERT. Alignment of human, M.musculus, M.auratus, R.norvegicus, X.laevis and A.thaliana TERT sequences (,,–46) was performed using the BLAST program. The symbol ‘+’ indicates non-identical conserved residues. Residues conserved in all vertebrate sequences are boxed in black. Residues boxed in gray are A.thaliana residues conserved with vertebrate sequences. Thick black lines above the protein sequence indicate the amino acids that are deleted in the hTERT variants. 1 = Δ936–945, 2 = Δ963–972, 3 = Δ993–1002, 4 = Δ1020–1029, 5 = Δ1047–1056, 6 = Δ1077–1086, 7 = Δ1108–1117, 8 = Δ1123–1132. The single amino acid substitutions L974A, L978A, L980A, T1030A, S1037A and S1041 are indicated by an asterisk. (B) A schematic illustration of hTERT. Mutations characterized in this study are indicated as broken (deletions) or white bars (substitutions) on the linear map of the hTERT C terminus. The numbering for each C-terminal variant indicates the amino acid positions of the deleted or substituted residues.
Figure 1
Figure 1
Map of the hTERT C terminus, location of C-terminal mutations, and sequence alignment of the C termini of vertebrate and A.thaliana TERTs. (A) Alignment of the C-terminal amino acid sequences of five vertebrate TERTs and A.thaliana TERT. Alignment of human, M.musculus, M.auratus, R.norvegicus, X.laevis and A.thaliana TERT sequences (,,–46) was performed using the BLAST program. The symbol ‘+’ indicates non-identical conserved residues. Residues conserved in all vertebrate sequences are boxed in black. Residues boxed in gray are A.thaliana residues conserved with vertebrate sequences. Thick black lines above the protein sequence indicate the amino acids that are deleted in the hTERT variants. 1 = Δ936–945, 2 = Δ963–972, 3 = Δ993–1002, 4 = Δ1020–1029, 5 = Δ1047–1056, 6 = Δ1077–1086, 7 = Δ1108–1117, 8 = Δ1123–1132. The single amino acid substitutions L974A, L978A, L980A, T1030A, S1037A and S1041 are indicated by an asterisk. (B) A schematic illustration of hTERT. Mutations characterized in this study are indicated as broken (deletions) or white bars (substitutions) on the linear map of the hTERT C terminus. The numbering for each C-terminal variant indicates the amino acid positions of the deleted or substituted residues.
Figure 2
Figure 2
Reconstitution of recombinant telomerases in an in vitro transcription/translation system and detection of telomerase activity using TRAP. Telomerase activity of C-terminal variants of hTERT expressed in RRL in the presence of hTR was detected using the PCR-based TRAP assay. Top panel: reconstituted telomerase activity of hTERT C-terminal mutants (Fig. 1). A PCR amplification control is indicated (IC). Bottom panel: expression of hTERT C-terminal mutants synthesized in RRL in the presence of [35S]methionine was detected by SDS–PAGE.
Figure 3
Figure 3
Detection of the catalytic activity of in vitro-reconstituted human telomerases using the direct primer extension telomerase assay. The telomerase activity of hTERT C-terminal variants expressed in RRL was detected using a non-PCR-based conventional assay. +6, +12, +18 and +24 refer to the first G in the telomeric sequence TTAGGG. The 3′-end-labeled biotinylated primer (TTAGGG)4 migrates as a 25mer [primer (P)+1], at the indicated position. (A) Upper panel: catalytic activity of RRL-expressed C-terminal deletion mutants and wild-type enzyme, and of native telomerase extracted from telomerase-positive HL-60 cells. Lower panel: a standard reaction assay was also performed with an [α-32P]-labeled (TTAGGG)4 primer added to the RRL for different periods of time. (B) Catalytic activity of C-terminal substitution mutants and wild-type enzyme.
Figure 3
Figure 3
Detection of the catalytic activity of in vitro-reconstituted human telomerases using the direct primer extension telomerase assay. The telomerase activity of hTERT C-terminal variants expressed in RRL was detected using a non-PCR-based conventional assay. +6, +12, +18 and +24 refer to the first G in the telomeric sequence TTAGGG. The 3′-end-labeled biotinylated primer (TTAGGG)4 migrates as a 25mer [primer (P)+1], at the indicated position. (A) Upper panel: catalytic activity of RRL-expressed C-terminal deletion mutants and wild-type enzyme, and of native telomerase extracted from telomerase-positive HL-60 cells. Lower panel: a standard reaction assay was also performed with an [α-32P]-labeled (TTAGGG)4 primer added to the RRL for different periods of time. (B) Catalytic activity of C-terminal substitution mutants and wild-type enzyme.
Figure 4
Figure 4
Average type I processivity values for wild-type and C-terminal mutant telomerases reconstituted in RRL. Processivity values were calculated as described in Materials and Methods. Each plot depicts the processivity of wild-type and mutant enzymes at a specific position within the telomerase repeat product. +1 refers to the second G in the telomeric repeat TTAGGG, +2 to the third G, +3 to the first T, +4 to the second T and +5 to the A. Mutants with significantly different processivity values compared with wild type (P < 0.01) are indicated by *.
Figure 5
Figure 5
Physical association of C-terminal hTERT mutants with GST–hTERT in vitro. GST–hTERT and C-terminal mutants were independently synthesized in RRL in the absence of hTR. Immunopurified GST–hTERT was mixed with partially purified [35S]methionine-labeled hTERT or C-terminal mutants. Co-precipitated 35S-labeled hTERT and immunoprecipitated GST–hTERT proteins were detected by SDS–PAGE/autoradiography (top panel) and by western blot analysis (bottom panel), respectively. Five percent of input proteins and 50% of immunoprecipitated proteins were loaded (top panel). Control reactions were performed in the absence of antibody (lane 19) or GST–hTERT (lane 20).
Figure 6
Figure 6
Inactive C-terminal mutants cannot functionally complement an inactive RT domain mutant to reconstitute telomerase activity. GST–hTERT D868N and C-terminal mutants of hTERT were co-synthesized in RRL in the presence of hTR and [35S]methionine. Telomerase activity was measured by TRAP assay (top panel). The Δ150–159 mutant (lane 2) is an inactive N-terminal hTERT mutant that functionally complements GST–hTERT D868N to reconstitute telomerase activity (17). The W547A mutant is an inactive N-terminal hTERT mutant (lane 3) that does not functionally complement GST–hTERT D868N to reconstitute telomerase activity (17). GST–hTERT D868N and hTERT mutants were detected by SDS–PAGE/autoradiography (bottom panel).

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