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DNA DYNAMICS AND CHROMOSOME STRUCTURE

Identification of Kluyveromyces lactisTelomerase: Discontinuous Synthesis along the 30-Nucleotide-Long Templating Domain

Tracy Boswell Fulton, Elizabeth H. Blackburn
Tracy Boswell Fulton
Departments of Microbiology and Immunology & Biochemistry and Biophysics, University of California, San Francisco, San Francisco, California 94143-0414
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Elizabeth H. Blackburn
Departments of Microbiology and Immunology & Biochemistry and Biophysics, University of California, San Francisco, San Francisco, California 94143-0414
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DOI: 10.1128/MCB.18.9.4961
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  • Fig. 1.
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    Fig. 1.

    Identification of K. lactis telomerase activity in vitro. (A) Schematic of K. lactis telomerase RNA (TER1) template region and the expected alignment of the primers KL22(17) and KL12(17). Arrowhead indicates the 3′ end position of correspondingly marked products in panels B and C. The boxed template residue corresponds to the site of the C-to-A mutation in theTer1-SnaBI strain examined in panel C. (B) Telomerase reactions were carried out with primers KL22(17) (lanes 1 to 5) and KL12(17) (lanes 6 to 8) as described in Materials and Methods, with the following changes: lane 2, pretreatment of extract with proteinase K (PK; 0.5 mg/ml) for 5 min at 25°C; lanes 3 and 7, pretreatment of extract with RNase A (10 μg/ml) at 25°C for 5 min (lane 3), followed by an additional 5-min incubation with 50 U of RNasin and 1 mM DTT; lane 4, pretreatment of extract with 50 U of RNasin and 1 mM DTT at 25°C for 5 min, followed by RNase A (10 μg/ml) for an additional 5 min; lane 5, no input primer; lane 8, ddATP substituted for dATP, with chain termination product marked. Reactions in lanes 6 to 8 were performed with 7.5 μM [α-32P]dTTP (400 Ci/mmol) as the radioactive label. The primer +1 position for terminal transferase-labeled KL22(17) is shown on the lower left side of lane 1. Product positions up to +8 are marked, but +8 products were visible only on longer gel exposures. Terminal transferase-labeled KL12(17) primer is in the lane marked M. Product positions up to +18 are marked, but +18 products were visible only on longer gel exposures. The high-molecular-weight products at the top of the gel were, as described previously for comparable S. cerevisiae extracts (3, 33), independent of RNase pretreatment or telomerase RNA as described below and were therefore not attributable to telomerase activity. Also, the diffuse band between +1 and +2 in lanes 1 to 5 was not produced by telomerase, as it formed independent of primer and was insensitive to RNase A and proteinase K. Std rxn, standard reaction. (C) Reactions with DEAE-fractionated extracts from wild-type (wt; lanes 1 to 7) and Ter1-SnaBI (lanes 8 to 14) strains were carried out with primer KL22(17) as described in Materials and Methods, with the following changes: lanes 1 and 8, pretreatment with RNase A as described above; lanes 3 and 10, no input primer; lanes 4 to 6 and 11 to 13, substitution of each indicated ddNTP for its partner dNTP; lanes 7 and 14, [α-32P]dGTP as the sole dNTP substrate. Shaded lanes highlight nucleotide incorporation differences between the wild-type and mutant extracts. Terminal transferase-labeled KL22(17) primer (lane M) marks the primer +1 position. The nucleotides predicted to be incorporated by wild-type and Ter1-SnaBI telomerase are marked on the left and right, respectively. (D) Reactions with DEAE-fractionated extracts from wild-type (lanes 1, 2, and 5) andter1-Δ7 (lanes 3, 4, and 6) were carried out with primer KL22(17) (lanes 1 to 4) and primer KL12(12) (lanes 5 and 6). Extract in lanes 2 and 4 was pretreated with RNase A as described above.

  • Fig. 2.
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    Fig. 2.

    Kinetics of K. lactis telomerase activity. Reactions were performed with wild-type DEAE-fractionated extract and primer KL22(17). (A) Plot of product yield from reactions containing 0, 2, 4, 8, or 10 μl of wild-type DEAE-fractionated cell extract. The sums of the +1 to +7 products for each reaction were quantified and are presented as percentages of the yield with 10 μl of extract. (B) Plot of product yield from reactions containing 0, 0.125, 0.25, 0.5, and 1 μM primer. Product yield was quantified as in panel A. (C) Reaction with 10 μl of extract and 1 μM primer incubated for various time periods. Products from a 45-s reaction followed by a 24-min chase with 100 μM unlabeled dGTP are shown in lane P/C. Any products resulting from translocative synthesis would have appeared in the top quarter of the gel region shown. (D) Plot of product yield from reactions shown in panel C. The sums of the +5 and +6 (marked by arrowhead) products were quantified from a scanned autoradiogram, using the program NIH Image, and the results are presented as percentages of the yield at 3 min.

  • Fig. 3.
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    Fig. 3.

    Association of K. lactis telomerase with elongation products. An in vitro telomerase reaction with primer KL13(12) was performed, and then the mixture was size fractionated on Sephacryl S-300 as described in Materials and Methods. (A) Aliquots of each fraction were separated on a native gel composed of 3% polyacrylamide (80:1 acrylamide/bisacrylamide) and 0.6% agarose (lanes 1 to 10; numbers correspond to fraction numbers), along with a portion of the nonfractionated telomerase reaction (Load lane) and 10 μl of the partially purified K. lactis extract that was used in the reaction (Extract lane). The gel was transferred to a Hybond Plus membrane (Amersham) and hybridized to a mixture of two32P-labeled fragments of the TER1 gene that exclude the template region. (B) Aliquots of each fraction were separated on a denaturing 15% polyacrylamide (20:1 acrylamide/bisacrylamide)–8 M urea gel (lanes 1 to 10), along with a portion of the nonfractionated telomerase reaction (Load lane). Arrowhead corresponds to products described in Fig. 1B, and positions of mid-template and near-terminal products are bracketed. The upper portion of the panel shows the fractionation pattern of a telomeric 30-mer, 32P labeled by terminal transferase and loaded on the S-300 column with the completed telomerase reaction. This portion of the panel is from an exposure of the gel 30-fold longer than that used to show telomerase products. (C) Plot of the relative amounts of TER1 RNA (A) and telomerase products (B) recovered in each fraction. Products in each fraction were quantified and are represented as a percentage of the total collected.

  • Fig. 4.
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    Fig. 4.

    Primer-dependent stalling by K. lactistelomerase. (A) Schematic of results from assays using 12-mer oligonucleotides aligning at different positions along the template. Numbers next to primers reflect the positions of the primers’ 3′ end on the template, as indicated by numbers above the template. Product bands are denoted at their appropriate template positions with black dots. The size of each dot represents the relative intensity of signal from products stalled at each position, and shaded columns of dots indicate the two preferred regions of stalling. Asterisks above positions 18 through 22 and the arrowheads above positions 3R and 4R mark 3′-end positions of products shown in panel B. (B) Reactions using primers 12 to 20, as depicted in panel A. Since all primers are the same length but align stepwise across the template, elongation of primers to the same position resulted in products varied by 1 nt in length (compare positions of arrowheads and asterisks).

  • Fig. 5.
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    Fig. 5.

    Dependence of K. lactis telomerase stalling on nucleotide concentration. (A) Reactions using the primer KL13(12) and dGTP concentrations equaling 3.75, 5.63, 7.50, 9.38, 11.25, 18.75, 35.63, and 52.50 μM (each reaction mixture contains 50 μCi of [α-32P]dGTP and a variable amount of unlabeled dGTP). Products of particular interest are marked on the left side by their 3′-end positions. Asterisks and arrowheads mark products corresponding to those shown in Fig. 4. (B) Plot of telomerase products at various dGTP concentrations. At each concentration, products stalled at template positions 18, 19, 20, 21, 22, 3R, and 4R were quantified, corrected for specific activity differences, and divided by the number of dG residues incorporated into each product. Results for position 18 were omitted due to interference of the diffuse background band visible in the last two lanes, which corresponds to the non-telomerase-generated product seen in Fig. 1B, lanes 1 to 5. (C) A scaled-up reaction mixture with 3.75 μM [α-32P]dGTP was incubated for 1.5 min and then divided into three aliquots; one part was stopped (short pulse lane) with proteinase K and SDS (see Materials and Methods), the second aliquot was mixed with 100 μM unlabeled dGTP and incubated for an additional 23.5 min (long chase lane), and the third was incubated for a total of 25 min with no chase (long pulse lane). Asterisks and arrowheads mark mid-template and near-terminal products, respectively, and boxed asterisks show unchaseable products.

  • Fig. 6.
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    Fig. 6.

    Dependence of telomerase stalling on primer-template complementarity. Lanes 1 to 7, products from reactions with primers 1 to 7 as illustrated above (lane numbers correspond to the numbers at the left of the primers). All primers have 3′ ends aligning at template position 16. Primers’ amounts of template complementarity increase by 1 nt at a time from 12 to 16 nt (number above each primer). Note that the overall length of the primers also increases, and therefore the +1 position changes on the gel correspondingly. Primers numbered 6 and 7 are both 16 nt long, with 12 nt of complementarity to the template. The 4 nt at the 5′ end of primer 6 are the same as those of primer 5 but scrambled so that none of them are complementary to the template. The nucleotide composition of the 5′ end of primer 7 is nontelomeric (ATAT). Lanes 8 to 11, products from reactions with primers, as illustrated. Primers in this set share 3′ ends that align at position 22. Again, the numbers above each primer reflect their degree of complementarity to the template. Primers 10 and 11 are both 17 nt long, and both have 12 nt of complementarity to the template. The 5 nt at the 5′ end of primer 10 are as in primer 9 but scrambled (as with primer 6). The 5′ end of primer 11 is nontelomeric (ATATA). Asterisks and arrowheads correspond to products previously discussed.

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Identification of Kluyveromyces lactisTelomerase: Discontinuous Synthesis along the 30-Nucleotide-Long Templating Domain
Tracy Boswell Fulton, Elizabeth H. Blackburn
Molecular and Cellular Biology Sep 1998, 18 (9) 4961-4970; DOI: 10.1128/MCB.18.9.4961

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Identification of Kluyveromyces lactisTelomerase: Discontinuous Synthesis along the 30-Nucleotide-Long Templating Domain
Tracy Boswell Fulton, Elizabeth H. Blackburn
Molecular and Cellular Biology Sep 1998, 18 (9) 4961-4970; DOI: 10.1128/MCB.18.9.4961
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KEYWORDS

Kluyveromyces
telomerase

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