(Received for publication, June 28, 1995 )
From the
Extracts of Saccharomyces cerevisiae were shown to
support the elongation of oligodeoxynucleotides with telomere-like
sequences. The primer sequence specificity of this elongation activity,
its incorporation of dG and dT but not dA or dC from the corresponding
triphosphates, and its sensitivity to RNase A and RNase H are all
consistent with it being a telomerase. In contrast to the reported
properties of other telomerases, the presence of ATP enhances the
efficiency of initiation of the yeast enzyme and improves its
processivity. Hydrolysis of ATP appears to be unnecessary for the
observed effects, as the ,
-imido or the
-thio derivative
of ATP is nearly as effective.
Telomerase is a ribonucleoprotein complex that adds GT-rich repeats to oligomeric or polymeric deoxynucleotide primers with telomere-like sequences at their 3` ends(1) . Because the enzyme uses an integral RNA component as the template in the synthesis of the repetitive deoxynucleotide sequences, it can be considered a specialized reverse transcriptase. Telomerase is necessary for maintaining the length and integrity of telomeres, which in turn have a variety of important physiologic functions, including the stabilization of chromosome ends against illegitimate recombination, the repression of telomere-proximal genes, and the contribution to chromosome organization inside the nucleus(2) .
Telomerase has been identified in a number of organisms, including ciliated protozoa, mouse, and human(3, 4, 5) . The RNA component of the enzyme complex has been cloned from several protozoa (6, 7, 8, 9) and more recently from yeast(10) , and genes encoding two polypeptide components of Tetrahymena telomerase were recently cloned(11) . In addition to being characterized at the gene level, the relative abundance of the Tetrahymena enzyme has also led to its extensive biochemical characterization. Nucleotide addition by Tetrahymena telomerase can be processive or nonprocessive in vitro depending on reaction conditions, and under special conditions the enzyme can also exhibit nuclease activity(12) .
Because of the ease of genetic analysis and manipulation of the budding yeast, this unicellular eukaryote provides an ideal system for mechanistic and functional studies of cellular complexes. A number of yeast mutants that affect telomere length have been identified (13, 14, 15, 16, 17) , some of which may affect telomerase activity either directly or indirectly. The gene TLC1 encoding the RNA component of yeast telomerase has recently been cloned(10) . The EST1 gene product has also been postulated to be a protein component of yeast telomerase(18) , but direct evidence is lacking; the newly identified Tetrahymena telomerase polypeptides also appear to be unrelated to the EST1 protein in their amino acid sequences. Despite the rapid accumulation of information on yeast genes that affect telomeric sequences, study of yeast telomerase to date has been hampered by the lack of a suitable in vitro assay. We describe such an assay in this report and show that the yeast enzyme exhibits an ATP dependence that was unknown for telomerases previously characterized.
Figure 3: Dependence of the yeast telomerase activity on oligodeoxynucleotide primers. Standard telomerase reactions were carried out using the indicated primer substrates. No primer was included in the lane1 sample.
Figure 1:
Dependence of the yeast telomerase
activity on dNTP. Standard telomerase assay components except for the
following deoxynucleotide triphosphate substrates were used for the
reactions: lane1, 2.5 mM dGTP, 20 µCi
of [-
P]dTTP (3000 Ci/mmol); lane2, 20 µCi of [
-
P]dTTP; lane3, 2.5 mM each of dTTP and dGTP, 20
µCi of [
-
P]dATP (3000 Ci/mmol), and 20
µCi of [
-
P]dCTP (3000 Ci/mmol); lane4, 2.5 mM dTTP, 20 µCi of
[
-
P]dGTP (3000 Ci/mmol); lane5, 20 µCi of
[
-
P]dGTP.
Figure 2:
Sensitivity of the yeast telomerase
activity to ribonucleases. Telomerase reaction components except primer
and nucleotides were first mixed, and the following reagents were then
added to the samples that were analyzed in the lanes specified: lane1, TELCA (1.0 µg) and RNase
H (20 units); lane2, TELH1 (1.0 µg) and RNase H
(20 units); lane3, RNase A (0.03 µg) and
Inhibit-ACE (Inh, 2 units, 5 Prime 3 Prime, Inc.,
Boulder, CO); lane4, RNase A (0.03 µg).
Following incubations at room temperature for 20 min, 2 units of
Inhibit-ACE were added to the lane2 sample, and
nucleotides and primers (TEL2 for lanes1 and 2, TEL1 for lanes3 and 4) were
added to all the reactions to allow telomerase extension to take place.
The reaction products were analyzed as described under
``Experimental Procedures.''
To demonstrate a specific requirement of the yeast activity for TLC1 RNA, the RNA component of yeast telomerase(10) , we pretreated the telomerase fraction with oligodeoxynucleotides complementary to TLC1 RNA and RNase H. The primer TEL2, which has a shorter stretch of sequence complementary to the template RNA than TEL1, was used in these assays to minimize substrate-dependent degradation of TLC1 RNA. As shown in Fig. 2, RNase H completely abolishes telomerase activity in the presence of an 18-mer antisense oligonucleotide TELH1 (lane2), which is complementary to the template region of TLC1 but not in the presence of a noncomplementary oligodeoxynucleotide TELCA of the same length (lane1).
Figure 4:
Modulation of the yeast telomerase
activity by ATP and its analogs. A, standard telomerase
reactions were carried out using TEL1 as primer. The following
nucleotide triphosphates were included at 5 mM: lane1, none; lane2, ATP; lane3, AMPPNP; lane4, ATPS; lane5, dATP; lane6, dCTP. B,
standard telomerase reactions were carried out using TEL1 as primer,
and AMPPNP was included at the indicated µM concentrations.
We have identified and partially purified from yeast extracts an activity that fulfills several criteria for being a telomerase. In particular, the inactivation of the activity by RNase H in the presence of an antisense oligodeoxynucleotide complementary to the TLC1 template RNA indicates that primer elongation is specifically dependent on the RNA component of the yeast telomerase. Our finding therefore opens up the prospect for a combined biochemical and genetic analysis of yeast telomerase.
The yeast telomeric repeats are more irregular in sequence in comparison with repeats in many other organisms such as human and Tetrahymena(20) . In the case of the latter two enzymes, the regularity of the repeat and the presence of preferred pulse sites within them give rise to elongation products that are integral multiples of the repeat unit(3, 5) . Not surprisingly, such a pattern was not readily discernible in the yeast enzyme reaction products. Nevertheless, an interesting question with regard to the yeast enzyme is whether a unique sequence is generated in a single round of extension of a particular primer. It is evident that the yeast enzyme preferentially generates products of specific lengths. If this reflects preferential pulsing or termination by the enzyme at a particular template position, then our results are consistent with the hypothesis that a unique sequence is made. This can be confirmed by cloning and sequencing the in vitro products of the activity.
One unexpected finding in this work is the role of ATP in the modulation of yeast telomerase activity, which has not been previously observed in the characterization of other telomerases. Since ATP binding appears to suffice, the observed effects are unlikely to be due to a catalytic helicase that exposes single-stranded substrate for telomerase binding. ATP binding could, for example, trigger a conformational change in the telomerase ribonucleoprotein complex or its accessory protein(s) to lower the dissociation rate of the complex. In contrast to the yeast enzyme, human and Tetrahymena telomerases are processive in the absence of ATP(3, 5) , and the mouse enzyme reportedly acts distributively even in the presence of ATP(4) . It is possible that modulation of telomerase activity by ATP is unique to the yeast enzyme. However, in the case of the human enzyme, interpretation of the experimental data might have been complicated by the presence of dATP, which was needed as a substrate for the synthesis of human telomeric repeats(5) . As shown in this work, dATP is an effective substitute for ATP in maintaining the processivity of the yeast enzyme and thus might act in similar ways in the human enzyme-catalyzed reaction.
Note Added in Proof-Two papers have appeared after the submission of this communication (Lin, J.-J., and Zakian, V. A.(1995) Cell81, 1127-1135; Cohn, M., and Blackburn, E. H. (1995) Science269, 396-400 in which the detection of telomerase activity in yeast cell extracts or partially purified fractions was reported.