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Telomerase is a ribonucleoprotein enzyme, which maintains genome integrity in eukaryotes and ensures continuous cellular proliferation. Telomerase holoenzyme from the thermotolerant yeast Hansenula polymorpha, in addition to the catalytic subunit (TERT) and telomerase RNA (TER), contains accessory proteins Est1 and Est3, which are essential for in vivo telomerase function. Here we report the high-resolution structure of Est3 from Hansenula polymorpha (HpEst3) in solution, as well as the characterization of its functional relationships with other components of telomerase. The overall structure of HpEst3 is similar to that of Est3 from Saccharomyces cerevisiae and human TPP1. We have shown that telomerase activity in H. polymorpha relies on both Est3 and Est1 proteins in a functionally symmetrical manner. The absence of either Est3 or Est1 prevents formation of a stable ribonucleoprotein complex, weakens binding of a second protein to TER, and decreases the amount of cellular TERT, presumably due to the destabilization of telomerase RNP. NMR probing has shown no direct in vitro interactions of free Est3 either with the N-terminal domain of TERT or with DNA or RNA fragments mimicking the probable telomerase environment. Our findings corroborate the idea that telomerase possesses the evolutionarily variable functionality within the conservative structural context.
(a) Western blot analysis (with anti-HA antibodies) of TERT-HA protein levels in anti-HA precipitates prepared from the indicated strains. 1/2 portions of the IP sample obtained from 400 ml YPD cultures (OD 600 ~ 1) were used for analysis. Numbers below are quantifications of band intensities (mean ± SD); data collected from experiments with three independently grown cultures of each strain. (b) Expression levels of telomerase RNA (HpTER) and HpTERT mRNA in TERT-HA EST3 and TERT-HA ∆est3 strains as determined by quantitative RT-PCR. qPCR signals from test RNAs were normalized to HpU1 snRNA as described in METHODs. Data obtained using four RNA preparations are plotted in the diagram (mean ± SD). (c) anti-HA Western blot analysis shows comparable amount of TERT-HA protein in 1/10 portion of the IP sample obtained from 400 ml YPD culture (OD 600 ~ 1) of TERT-HA EST3 and in 1/2 portion of the IP sample obtained from 1,600 ml YPD culture (OD 600 ~ 1) of TERT-HA ∆est3. The equivalent amounts of IP samples were analyzed by primer extension assay (in d). (d) Telomerase activity assay of TERT-HA EST3 and TERT-HA ∆est3 IP samples (containing similar amount of TERT) with HD5 primer. Telomerase elongation products (up to + 9 nucleotides) are visible in TERT-HA EST3 sample, but not in TERT-HA ∆est3. "LC" loading control (a [γ-32 P]-labeled 13-mer oligomer). Data in (c and d) are representative of three independent experiments. Original (full lane view, no contrast adjustment) blots and gels from (a, c and d) are shown in Supplementary Figs. S14 and S15.
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
SCIENTIFIC REPORTS | (2020) 10:11109 | 
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Insights into the structure
andfunctionofEst3
from the Hansenula polymorpha
telomerase
Nikita M. Shepelev1,2,5,SoaS.Mariasina1,3,5,AlexeyB.Mantsyzov1,
AlexanderN.Malyavko1,SergeyV.Emov4,OlgaA.Petrova1,ElenaV.Rodina1,
Maria I. Zvereva1,OlgaA.Dontsova1,2*&VladimirI.Polshakov1*
Telomeraseisaribonucleoproteinenzyme,whichmaintainsgenomeintegrityineukaryotesand
ensurescontinuouscellularproliferation.Telomeraseholoenzymefromthethermotolerantyeast
Hansenula polymorpha,inadditiontothecatalyticsubunit(TERT)andtelomeraseRNA(TER),
containsaccessoryproteinsEst1andEst3,whichareessentialforinvivotelomerasefunction.Here
wereportthehigh-resolutionstructureofEst3fromHansenula polymorpha(HpEst3)insolution,as
wellasthecharacterizationofitsfunctionalrelationshipswithothercomponentsoftelomerase.The
overallstructureofHpEst3issimilartothatofEst3fromSaccharomyces cerevisiaeandhumanTPP1.
WehaveshownthattelomeraseactivityinH. polymorphareliesonbothEst3andEst1proteinsina
functionallysymmetricalmanner.TheabsenceofeitherEst3orEst1preventsformationofastable
ribonucleoproteincomplex,weakensbindingofasecondproteintoTER,anddecreasestheamount
ofcellularTERT,presumablyduetothedestabilizationoftelomeraseRNP.NMRprobinghasshown
nodirectinvitrointeractionsoffreeEst3eitherwiththeN-terminaldomainofTERTorwithDNAor
RNAfragmentsmimickingtheprobabletelomeraseenvironment.Ourndingscorroboratetheidea
thattelomerasepossessestheevolutionarilyvariablefunctionalitywithintheconservativestructural
context.
Abbreviations
CaEst1, CaEst3, CaTER Est1, Est3 proteins and TER from Candida albicans
HpTER, HpTERT, HpEst1, HpEst3 TER, TERT, Est1 or Est3 from Hansenula polymorpha
hPOT1, hTPP1 Human proteins POT1, TPP1
EMSA Electrophoretic mobility shi assay
HSQC Heteronuclear single quantum coherence spectroscopy
NOE Nuclear Overhauser eect
NOESY Nuclear Overhauser enhancement spectroscopy
R1 Longitudinal or spin–lattice relaxation rate
R2 Transverse or spin–spin relaxation rate
Rex Conformational exchange contribution to R2
RMSD Root-mean-square deviation
S2 Order parameter reecting the amplitude of ps-ns bond vector
dynamics
ScEst3, ScTERT Est3 or TERT proteins from Saccharomyces cerevisiae
SnTEBP TEBP protein from Sterkiella nova
OPEN
            
            
            
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           *email: dontsova@
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SOFAST HMQC Band-selective optimized ip angle short transient heteronuclear
multiple-quantum correlation experiment
TEN N-terminal domain of TERT
TERT Telomerase reverse transcriptase
TER Telomerase RNA
TERT, EST1, EST3 Genes encoding TERT, Est1, Est3 proteins
TERT-HA, Est1-HA, Est3-HA TERT, Est1 or Est3 proteins tagged with a hemagglutinin epitope (HA)
TERT-HA, EST1-HA, EST3-HA Strains expressing TERT (Est1, Est3) tagged with a hemagglutinin
epitope
τe Eective internal correlation time
τm Overall rotational correlation time
tert, ∆est1, ∆est3 Strains with TERT (EST1, EST3) gene deleted
Telomerase is an enzyme essential for the synthesis and replication of telomeres—nucleoprotein structures
capping the ends of eukaryotic chromosomes. Telomerase’s core enzyme is a reverse transcriptase containing
a protein catalytic subunit (TERT) and telomerase RNA (TER)13. Using a portion of TER as a template, TERT
elongates the 3-ends of telomeric single-stranded DNA, synthesizing a string of telomeric repeats4. e action of
telomerase compensates for the loss of telomeric DNA through successive cell divisions due to the end-replication
problem58. In human, most dierentiated somatic cells lack telomerase activity. In highly proliferative cells, e.g.
unicellular eukaryotes and embryonic cells of mammals, telomerase expression and activity is tightly controlled,
although the exact mechanisms of this regulation are poorly understood911 (see also12 for a recent review). e
malfunction of these mechanisms and up-regulation of telomerase is the leading cause of cell immortalization
in most types of cancer1316. However, the understanding of telomerase function and regulation remains a chal-
lenge due to limited amount of structural information on the organization of the telomerase complex and the
interactions of its components.
In vivo the function of telomerase, in addition to the core enzyme, requires accessory protein subunits,
which vary considerably from one species to another (reviewed in Ref.17), and their precise role in the regula-
tion of telomerase is still not completely understood. In budding yeast Saccharomyces cerevisiae such accessory
telomerase subunits are Est1, Est3, a heptameric ring of Sm proteins, yKu heterodimer, and a recently discov-
ered set of the Pop proteins (Pop1/Pop6/Pop7)1826. Sm7 binds near the 3-end of the TER and is required for
its stability and maturation21. yKu is implicated in the nuclear import of the telomerase RNA, and plays role
in the association of telomerase with telomeres1820. Pop1/Pop6/Pop7 stimulate association of TERT and Est1
with telomerase RNA invivo22,23. ey are dispensable for TERT activity invitro, and their precise role in the
regulation of telomerase is still not completely understood. Deletion of either EST1 or EST3 leads to gradual
telomere loss and loss of viability identical to the Est2 and TLC1 (homologs of TERT and TER in S. cerevisiae)
null mutants25, yet telomerase isolated from ∆est1 or ∆est3 strains was still active invitro27. Est1 contains an
RNA binding domain and interacts directly with a specic stem within telomerase RNA28,29. Est1 also directly
binds the telomeric ssDNA-binding protein Cdc13, and this interaction is crucial for telomerase recruitment
at telomeres3033. Apart from its recruitment function, Est1 is also required for loading Est3 into the telomerase
complex3436. Est3 is a small protein unable to bind telomerase RNA directly and has to rely on protein–protein
interactions to become a part of the telomerase holoenzyme. In addition to Est1, Est3 was shown to interact in
S. cerevisiae with Est2, specically with its N-terminal domain (TEN)35,3739. It was found that Est3 associates
with the preassembled Est1-TLC1-Est2 subcomplex with maximum binding observed only late in the cell cycle
(late G2/M), suggesting that Est3 loading is a highly regulated step during telomerase assembly35. However, the
mechanism of telomerase activation by Est3 association still remains unclear.
In other budding yeast species (Saccharomyces castellii and Candida albicans) the requirement for Est3 protein
for telomerase activity invitro appears to be more pronounced than in S. cerevisiae38,40. Interestingly, in the case
of C. albicans, the eect of EST3 deletion depends on the primer used in the telomerase assay40. Whether these
observations point to mechanistic distinctions in Est3 functions in dierent species is yet to be determined.
However, deletion of EST1 in C. albicans leads to an almost identical defect41. Remarkably, in the est3-null strain,
CaEst1 loses the ability to bind telomerase RNA (the same is true for the est1-null strain and CaEst3), further
stressing dierences in telomerases from diverse species40.
e structure of S. cerevisiaes Est3 protein (lacking 12N-terminal amino acids) was obtained using a strategy
that combines minimal NMR experimental data with Rosetta de novo structure prediction algorithm42. is
study revealed that ScEst3 is an OB-fold, as was predicted earlier43; however, the experimentally obtained struc-
ture diers signicantly from the predicted model. e unbiased mutagenesis of every surface residue dened
a surface which is important for the association of ScEst3 with the telomerase complex42. Although the human
telomerase complex does not contain an obvious homologue of Est3, one human telomeric protein—TPP1—
has an OB-fold domain44 structurally very similar to ScEst3. hTPP1 was shown to be important for telomerase
recruitment to telomeres via interaction with the TEN domain of human TERT, and the residues responsible
for the interaction cluster in a single surface (dubbed the TEL patch)45,46. Most strikingly, the identied surface
of ScEst3 almost coincides with the TEL patch on hTPP1, suggesting the functional importance of the Est3/
TPP1—TEN interaction for telomerase action42.
Previously we published the 1H, 13C and 15N resonance assignments of HpEst347. Here we report the high-
resolution solution structure of HpEst3 along with genetic and biochemical characterization of this protein. We
believe that this information is essential for a deeper understanding of the mechanism of telomerase function
and regulation.
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Results
Hansenula polymorphaEst3 is essential for telomeraseactivity. To conrm that the identied
HpEst3 homologue is required for telomerase action we constructed a H. polymorpha strain (∆est3) with Est3
open reading frame substituted for HpLEU2 marker. ∆est3 strain was propagated in liquid YPD medium for sev-
eral days. As it was described for knock-outs of other H. polymorpha telomerase components (HpTER, HpTERT
and HpEst1)48,49, ∆est3 cells rapidly lost telomeric DNA and exhibited greatly reduced viability at the earliest
passages (Fig.1a, b). A population of “survivors” subsequently emerged, which maintain their telomeres pre-
sumably via recombination.
To isolate telomerase and to investigate the possibility that Est3 is necessary for telomerase activity invitro,
we also constructed a strain expressing HpTERT tagged with a hemagglutinin epitope (TERT-HA). e tagged
TERT behaves as the native protein, as judged by the telomere length analysis (Fig.1c). Extract prepared from
the TERT-HA EST3 strain was incubated with anti-HA agarose, and telomerase activity precipitated on the
beads was assessed by its ability to elongate 13-mer oligonucleotide HD5 in the presence of radiolabelled dGTP.
We detected several elongation products (up to + 9) expected from the HpTER template sequence (Fig.1d, e).
Surprisingly, the amount of TERT-HA precipitated from the TERT-HA ∆est3 strain was reduced ~ 25-fold
compared to the parental TERT-HA EST3 strain (in which the EST3 gene is intact), suggesting that the Est3
protein is required for the normal accumulation of TERT protein in H. polymorpha cells (Fig.2a). We did not
detect any reduction in TERT mRNA abundance upon deletion of the EST3 gene; therefore, Est3 inuences
either translation process or TERT protein stability (Fig.2b). Of note, HpTER RNA levels are identical in EST3
and ∆est3 strains; therefore, not all telomerase subunits are downregulated aer Est3 loss (Fig.2b). Taking into
account this eect we compared telomerase preparations from EST3 and ∆est3 containing a similar amount of
TERT-HA protein in primer elongation assay. Despite the presence of TERT at comparable levels in both prepa-
rations (Fig.2c), we did not detect any nucleotide addition by the telomerase isolated from the TERT-HA ∆est3
strain (Fig.2d). Unfortunately, we could not reliably determine the HpTER content of the two samples, because
the amount of HpTER co-puried with TERT-HA from the ∆est3 strain was very small (close to background
levels), leading to large experimental error. Collectively, results from this section show that Est3 is an essential
protein for telomerase action in H.polymorpha and is required for the normal accumulation of TERT protein.
Figure1. (a) Spot assay. EST3 (wild type) and ∆est3 (two clones isolated aer transformation) strains were
passaged in liquid SC + LEU (EST3) or SC-LEU (∆est3) medium. Cell culture aliquots (and three serial tenfold
dilutions) from the indicated passage were spotted onto a YPD plate, grown for 2days and photographed. (b)
Southern blot analysis of terminal restriction fragments from the indicated strains. Genomic DNA was isolated
from cells aer each passage (passage number is shown under each lane). (c) Same as (b), however DNA
used for this analysis was isolated from yeast cultures aer 10 restreaks on agar plates (~ 200 generations). (d)
Telomerase activity assay of the TERT-HA EST3 IP sample [isolated from 400ml of YPD culture (OD600 = 1)]
with HD5 primer, radiolabeled dGTP and unlabeled dTTP, dCTP, dATP. Positions of + 1, + 4, + 7 and + 9
elongation products are indicated; faint bands longer than + 9 most likely correspond to products resulting from
translocation of the + 8 and the second round of the template copying (type II translocation). “LC” loading
control (a [γ-32P]-labeled 13-mer oligomer). (e) Schematic of HD5 primer alignment along the template region
of the HpTER (nucleotides 170–187). Nucleotides added by telomerase in the invitro assay are in bold. Original
(full lane view, no contrast adjustment) Southern blots and gels from (b, c and d) are shown in Supplementary
Fig.S14.
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Est3structuredetermination. e NMR signal assignments of the HpEst3 protein have been reported
earlier47. A family of 20 NMR structures was determined using 2,490 experimental restraints measured at 298
and 303K (see Table1 for details). is work made use of standard double and triple resonance NMR methods
applied to 15N and 15N/13C labeled samples of HpEst3. For most of the protein residues, the number of NOEs per
residue is between 20 and 40 (Supplementary Fig.S1). is number is less for the N and C-terminal residues and
residues from several protein loops. e protein consists of the structured β-barrel core (residues 22–172) and
disordered N and C-terminal tails (residues 1–22 and 173–178) (see Fig.3b). Five β-strands surrounded by six
α-helices and several loops form the protein core (Fig.3a). e protein core is well structured: root-mean-square
deviation (RMSD) of the coordinates of heavy backbone atoms of the residues 22–172 in the family of 20 NMR
conformers is 0.87 ± 0.14Å (Table1). In the Ramachandran plot analysis (Supplementary Fig.S2), 87% of the
residues in the whole NMR family were found in the most favored regions and none in the disallowed regions.
e structure of HpEst3 adopts a classic OB-fold topology50,51 similar to that of Est3 from Saccharomyces cer-
evisiae42 or human TPP144,52 (see “Discussion”). e family of HpEst3 structures and experimental restraints used
in solution structure calculations have been deposited in the Protein Data Bank under accession number 6Q44.
Proteinbackbonedynamics. Dynamic properties of the HpEst3 backbone in ps-ns and ms time scales
were studied using the 15N relaxation parameters for the amide 15N nuclei. Longitudinal (R1) and transverse
(R2) relaxation rates measured at 298K and heteronuclear 15N-1H Overhauser eects are shown on Fig.4a–c.
Model-free analysis of the experimental data allowed us to obtain values of the order parameter S2 (Fig.4d),
which reects the amplitude of ps-ns amide bond vector dynamics and chemical exchange contribution to the
transverse relaxation rate Rex (Fig.4e), manifesting protein motions occurring in the ms time scale. e value
of the correlation time of protein tumbling τm calculated from the experimental R2/R1 ratios is 13.6 ± 2.0 ns.
Applying models of anisotropic motions only slightly improves the data t. us, for the axial anisotropy model,
the ratio of the principal axes of the anisotropy tensor (D/D) is less than 1.2. erefore, model-free analysis
Figure2. (a) Western blot analysis (with anti-HA antibodies) of TERT-HA protein levels in anti-HA
precipitates prepared from the indicated strains. 1/2 portions of the IP sample obtained from 400ml YPD
cultures (OD600 ~ 1) were used for analysis. Numbers below are quantications of band intensities (mean ± SD);
data collected from experiments with three independently grown cultures of each strain. (b) Expression levels of
telomerase RNA (HpTER) and HpTERT mRNA in TERT-HA EST3 and TERT-HA ∆est3 strains as determined
by quantitative RT-PCR. qPCR signals from test RNAs were normalized to HpU1 snRNA as described in
METHODs. Data obtained using four RNA preparations are plotted in the diagram (mean ± SD). (c) anti-HA
Western blot analysis shows comparable amount of TERT-HA protein in 1/10 portion of the IP sample obtained
from 400ml YPD culture (OD600 ~ 1) of TERT-HA EST3 and in 1/2 portion of the IP sample obtained from
1,600ml YPD culture (OD600 ~ 1) of TERT-HA ∆est3. e equivalent amounts of IP samples were analyzed by
primer extension assay (in d). (d) Telomerase activity assay of TERT-HA EST3 and TERT-HA ∆est3 IP samples
(containing similar amount of TERT) with HD5 primer. Telomerase elongation products (up to + 9 nucleotides)
are visible in TERT-HA EST3 sample, but not in TERT-HA ∆est3. “LC” loading control (a [γ-32P]-labeled 13-mer
oligomer). Data in (c and d) are representative of three independent experiments. Original (full lane view, no
contrast adjustment) blots and gels from (a, c and d) are shown in Supplementary Figs.S14 and S15.
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of relaxation data has been carried out in the assumption of isotropic motion. As expected, residues from the
unstructured N- and C-terminal tails undergo fast internal motions resulting in small values of S2. ere are
several loops which have increased internal mobility in the ps-ns time scale (Fig.5a). Among them are the loop
L12 (residues 50–59), the loop L45, which separates helix α4 and strand β5 (residues 118–131), and the residues
144–161 between strand β5 and helix α6, including helix α5 in the middle (see Fig.4d). However, the most
interesting feature of Est3 is the protein dynamics in ms time scale, associated with an exchange between two or
more conformations (Fig.5b). Almost all protein residues of the structured protein core are involved in such a
conformational exchange (Fig.4e). e values of Rex for most of the amide 15N nuclei are between 2 and 10Hz
and maximum values observed for the residues 53, 64, 97, 131, 166, 167. ese correspond to the fragments of
helices α2, α3 and α6 and loops L12, L23 and L45 (Fig.5b). Conformational broadening of the Est3 resonances is
clearly observed in NMR spectra recorded at lower temperature (Supplementary Fig.S3). A decrease in tempera-
ture of only 10–15°C results in a signicant broadening of all the Est3 resonances except the unstructured tails.
Many signals are disappearing in the spectra measured at 288K. is indicates that 288K just slightly exceeds
the coalescence temperature of the chemical exchange for the most such signals.
ProbingtheinteractionofEst3 withcomponentsof telomeraseandtelomeres. e possible
interaction of HpEst3 with partners in the formation of the telomerase complex was probed by heteronuclear
NMR spectroscopy. e interactions of Est3 with TEN, as well as with the single-stranded DNA fragments
corresponding to telomeric repeats, RNA constructs, corresponding to TER fragments, and RNA–DNA heter-
oduplexes were investigated (see Supplementary TableS1). Interactions were monitored by the changes of 1H
and 15N chemical shis of HpEst3 upon the increase of the concentration of each tested binding partner (see
Supplementary Figs.S4–S11). To study the binding of HpEst3-TEN, two complementary experiments were car-
ried out, one of which tested a change in the chemical shis of HpEst3, and the other—TEN. In none of the cases
specic binding of HpEst3 with nucleic acids or TEN has been observed. When HpEst3 interaction with ssDNA
fragment that corresponds to four telomeric repeats (G4) was monitored, protein binding to DNA was observed,
but it was not specic. With an addition of long single-stranded DNA to protein, a signicant broadening of the
HpEst3 signals was observed, indicating the formation of high molecular weight complexes (Supplementary
Fig.S6). However, no noticeable changes in chemical shis characteristic of specic binding occurs.
We also tested the ability of Est3 to bind DNA in electrophoretic mobility shi assay, using uorescently
labeled DNA oligonucleotide containing four telomeric repeats (fG4) as a probe. We did not observe any bands
corresponding to Est3-fG4 complex even at relatively large concentrations of protein and DNA (10μM and 1μM,
respectively) (Supplementary Fig.S12a). Small portion of uorescent signal remained trapped in the gel wells,
which probably correspond to non-specic aggregation observed in the NMR experiment.
Association of Est3 protein with telomerase RNA is Est1-dependent, butTERT-independ-
ent. Our inability to detect interaction between HpEst3 and HpTEN invitro prompted us to check which
components are necessary for recruitment of Est3 into the telomerase complex in H. polymorpha. To monitor
Table 1. Statistics for the ensemble of the calculated 20 structures of the HpEst3. No NOE or dihedral
angle violations are above 0.5Å and 5° respectively. a < S > is the ensemble of 20 nal structures; Srep is the
representative structure, selected from the nal family on the criteria of having the lowest sum of pairwise
RMSD for the remaining structures in the family.
A. Restraints used in the structure calculation
Total NOEs 2,262 Total dihedral angles 228
Long range (|i−j|> 4) 443 Phi (ϕ)116
Medium (1 < |i−j|≤ 4) 210 Psi (ψ) 112
Sequential (|i−j|= 1) 568
Intraresidue 1,041
B. Restraint violations and structural statistics (for 20 structures)
Average RMSD < S > aSrep
From experimental restraints
Distance (Å) 0.042 ± 0.002 0.041
Dihedral (°) 0.269 ± 0.053 0. 253
From idealized covalent geometry
Bonds (Å) 0.0021 ± 0.0001 0.0019
Angles (°) 0.3540 ± 0.0102 0.3500
Impropers (°) 0.2380 ± 0.0145 0.2420
Ramachandran plot statistics
% of residues in most favorable region of Ramachandran plot 81.3 87.0
% of residues in disallowed region of Ramachandran plot 0.0 0.0
C. Superimposition on the representative structure (Å)
Backbone (C, Cα, N) RMSD over the structured protein core (residues 22–172) 0.87 ± 0.14
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Est3-HpTER association we constructed a strain (EST3-HA) expressing tagged Est3 from its native genomic
locus. e EST3-HA allele is functional: no major telomere shortening was observed in the strain (Fig.1c), and
HpTER robustly co-elutes with Est3 following immunoprecipitation (IP) on anti-HA agarose (Fig.6a). Notably,
the amount of HpTER coprecipitated with Est3-HA is not inuenced by the absence of the TERT gene (Fig.6a).
In sharp contrast, deletion of the EST1 gene completely abolishes the HpTER signal in the Est3-HA IP sample
(Fig.6a). e levels of isolated Est3-HA are identical for all three strains (EST3-HA, EST3-HAtert, and EST3-
Figure3. e solution structure of the HpEst3. (a) e topology of the secondary structure elements of the
HpEst3 protein. (b) e stereo view of the ensemble of the nal 20 calculated structures. Images were made
using PyMOL v. 2.3 (Schrodinger, LLC).
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HAest1) (Fig.6b). us, in H. polymorpha, recruitment of Est3 into the telomerase complex relies on the
presence of Est1 protein, rather than its direct interaction with the TEN domain of TERT as it was observed in
other species.
Similarly, we investigated the possibility that Est3 is required for Est1-HpTER interaction, using an EST1-
HA strain [again, functionality of the tagged protein was conrmed by the telomere length analysis (Fig.1c)].
Indeed, we found that Est1-HpTER association is eectively abolished in the absence of Est3 (Fig.6c, d). Deletion
Figure4. e relaxation parameters of the amide 15N nuclei of each residue of the HpEst3, measured at 16.3T
(700MHz proton resonance frequency) and 298K. (a) e heteronuclear 15N,1H-steady-state NOE values. (b)
e longitudinal relaxation rate R1 (s−1). (c) e transverse relaxation rate R2 (s−1). (d) e order parameter S2,
determined by model-free analysis. (e) Chemical exchange Rex contributions to the transverse relaxation rates
(s−1).
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of TERT also reduces the amount of HpTER co-immunoprecipitated with Est1-HA, however this eect is less
pronounced than in the case of EST3 deletion (Fig.6c). erefore, Est1 and Est3 proteins rely on each other to
bind HpTER.
e ~ tenfold dierence in co-IP HpTER between the Est1-HA and Est3-HA WT samples (Fig.6a, c) appar-
ently contradicts this latter statement, suggesting that a large portion of cellular Est3 may associate with Est1-free
(and, potentially, TERT-free) HpTER. However, we should note that the two experiments shown in Fig.6a, c were
performed using dierent batches of the anity resin, which may translate to dierent IP eciencies between
experiments. Also, the observed eect may result from variations in epitope accessibility. Indeed, when we
compared HpTER co-IP eciencies between Est1-HA and Est3-HA WT strains processed in parallel, we found
reduced Est3-HA/Est1-HA ratio (~ threefold, Supplementary Fig.S13a). Moreover, Est3-HA sample contained
more telomerase activity, compared to Est1-HA, indicating that telomerase complex is more eciently immu-
noprecipitated via Est3-HA (Supplementary Fig.S13b, c).
Given this interdependence of Est1 and Est3 for interaction with telomerase RNA, we tested whether the dele-
tion of EST1 would have any eect on the TERT protein levels, as we observed for the est3-null strain (Fig.2a).
Remarkably, the amount of TERT-HA is greatly reduced in the TERT-HA ∆est1 strain and indistinguishable from
the TERT-HA ∆est3 strain [both in whole-cell extracts and in the eluates aer immunoprecipitation on anti-HA
agarose (Fig.6e, f)]. Moreover, telomerase from the TERT-HA ∆est1 strain was decient in primer elongation
invitro (Fig.6g, h), further emphasizing the functional link between Est1 and Est3 in H. polymorpha. Consistent
with the absence of Est1 in telomerase preparation from the TERT-HA ∆est3 strain, addition of recombinant Est3
did not restore invitro defect of the TERT-HA ∆est3 telomerase (Fig.6i). Although, since we could not reliably
determine the level of telomerase RNA in the TERT-HA ∆est3 sample, this result may be also explained by the
absence of HpTER. Finally, we discovered that deletion of HpTER leads to the same (if not stronger) defect in
the TERT-HA protein accumulation (Fig.6j, k), suggesting that TERT stabilization by Est1 and Est3 may be
mediated through HpTER.
Discussion
As expected, we found that Est3 protein is an essential subunit of H. polymorpha telomerase complex, required
for telomeric DNA addition invivo. We also showed that telomerase from the ∆est3 strain is defective in the
primer elongation invitro. e requirement of Est3 for invitro telomerase activity has also been observed in
other yeast systems3840, although the degree of this requirement varies; and the nature of the Est3’s eect on
nucleotide addition by telomerase still remains elusive.
e determined high-resolution structure of HpEst3 showed high level of similarity with previously obtained
structures of hTPP144 and ScEst342 (Fig.7). ey all have an OB-fold typical for oligonucleotide/oligosaccharide
binding proteins42,53. e OB fold domains are widespread in proteins. Due to their high structural plasticity, OB
fold modules may be adapted to various functionalities. e most extensively characterized is the ssDNA-binding
Figure5. A cylindrical ribbon representation of the backbone of the C domain of the HpEst3. e variable
radius/thickness of the cylinder is proportional to the dynamic properties. (a) Motions in the ps to ns time scale.
ickness of the cylinder is proportional to the value of (1−S2); the minimal thickness corresponds to the value
S2 = 1, the maximum to S2 = 0.15. (b) Motions in the ms time scale. ickness of the cylinder is proportional to
the value of Rex; the minimal thickness corresponds to the value Rex = 0, the maximum to Rex = 20. Figure was
made using the Insight II v. 2000 soware (Molecular Simulations Inc.).
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ability of OB-fold proteins; among other known functionalities are their interactions with RNA and proteins,
including other OB fold units. OB fold domains were found in a number of telomere-binding proteins from
various species, where they demonstrate all these modes of interaction. e structural core of OB-fold proteins is
composed of conserved beta-strand elements β1–β5 forming the two anti-parallel beta-sheets, connecting loop
elements L12, L23, and L45 of variable length, and optional α-helices, the most conserved of which is a C-terminal
helix (C-helix). Analysis of the architecture of telomeric OB fold-containing proteins53 suggests that the length
and position of connecting elements and alpha-helices may reect the interaction preferences of a particular OB
fold module. Figure8 shows the examples of the ssDNA- and protein-binding telomeric proteins5456 with the
focus on their interaction interfaces and the position of the connecting element L45. Typical for these proteins is
Figure6. (a) Analysis of HpTER association with Est3-HA. Quantitative RT-PCR analysis of the HpTER
co-precipitated on anti-HA agarose aer incubation with extracts from the indicated strains. Small nuclear RNA
HpU1 was also analyzed as a negative control. Data represented as percentage of input RNA (mean ± SD). (b)
Western blot analysis (with anti-HA antibodies) of Est3-HA protein levels in anti-HA precipitates prepared from
the equal amounts of cultures of the indicated strains. Numbers below are quantications of band intensities
(mean ± SD) calculated using values from three biological replicates. “nd”—not determined. Extracts prepared
from three independently grown cultures of each strain were utilized for the experiments in (a) and (b). (c) and
(d) same as (a) and (b), respectively, only with Est1-HA protein. (e) same as (b) but with TERT-HA protein. 1/2
portions of the IP sample obtained from 400ml YPD cultures (OD600 ~ 1) were used for analysis. (f) Anti-HA
Western blot analysis of TERT-HA protein levels in extracts prepared from the indicated strains (upper panel).
Ponceau S-stained membrane served as a loading control (lower panel). Equal amounts of input samples were
used for analysis. Numbers below the blot are quantications of band intensities (mean ± SD) calculated using
values from three biological replicates. “nd”—not determined. (g) Telomerase activity assay of TERT-HA EST1
and TERT-HA ∆est1 IP samples with HD5 primer. “LC” loading control. 1/10 portion of the IP sample obtained
from 400ml YPD culture (OD600 ~ 1) of TERT-HA EST1 and in 1/2 portion of the IP sample obtained from
1,600ml YPD culture (OD600 ~ 1) of TERT-HA ∆est1 were used for the experiment. (h) Western blot analysis
of samples from (g). (i) same (g) but with TERT-HA EST3 and TERT-HA ∆est3 IP samples. Telomerase on
beads was pre-incubated with recombinant Est3 (rEst3) (1 or 10μM) where indicated. (j) same as (e) but
with TERT-HA ∆ter strain. (k) same as (f) but with TERT-HA ∆ter strain. Original (full lane view, no contrast
adjustment) blots and gels from (b, dk) are shown in Supplementary Fig.S16.
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a "canonical" binding surface capable of interacting with ssDNA or extended peptide fragments, dened by the
loops L45 and L12 from above and below. In addition, OB modules obviously can interact with protein molecules,
including other proteins or other OB modules of the same protein. Unlike the classical oligonucleotide-binding
interface, this interaction surface can vary in dierent OB fold proteins. One of such surfaces is located below the
β-barrel and is mediated with the L34 loop which in dierent OB fold proteins may include dierent secondary
structure elements. It appears that, in some cases, binding of the two protein molecules or subunits is necessary
for the tight interaction between OB fold domain and the oligonucleotide partner, and, vice versa, binding of
DNA is necessary to form the binding interface for the protein partner. Such cooperativity can be seen in the
structure of the SnTEBPα/β-DNA complex from Sterkiella nova (Fig.8b) where the protein-DNA interaction
site is formed in the α/β subunit interface. OB fold modules within the human heterodimer hPOT1/hTPP1 are
structurally similar to the SnTEBPα/β complex. Of this pair, only hPOT1 is capable of binding ssDNA directly,
Figure7. Structures (top) and Coulomb charge distribution over the molecular surfaces (bottom) of Est3 from
(a) Saccharomyces cerevisiae (PDB ID 2M9V42), (b) Hansenula polymorpha (PDB ID 6Q44, this work), and (c)
human TPP1 (PDB ID 2I4644). Images were made using PyMOL v. 2.3 (Schrodinger, LLC).
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but formation of a complex hPOT1/hTPP1 tightens this interaction tenfold44,57, which may indicate the analo-
gous structural interdependence. In the budding yeasts, neither structural homologs of POT1 or TEBPα nor
interaction between Est3 and nucleic acid partners was found. However, Est3 in S. cerevisiae interacts directly
with Est1 and with TERT, both of which bind telomerase RNA. ese interactions may also be cooperative due
to the plasticity of OB fold units.
In spite of the overall structural similarity, there are important dierences between HpEst3, ScEst3, and
hTPP1. First of all, unlike hTPP1 and many other OB fold proteins which have a short L45 loop that does not
obstruct the classical ligand-binding interface, the L45 loops in both yeast proteins (HpEst3 and ScEst3) are
signicantly larger (Fig.7). is prevents the interaction with potential ligands at the canonical interface without
a prior change in the loop conformation. Such conformational transitions are oen observed when OB-fold pro-
teins interact with DNA or RNA58. Dynamic properties of HpEst3 determined by 15N relaxation measurements
indicate the occurrence of conformational rearrangements of the protein backbone around the canonical OB-fold
ligand-binding interface (Fig.5b). erefore, we cannot exclude the possibility of exposing the ligand-binding
interface in HpEst3 due to the conformational rearrangements of the protein molecule.
Another signicant dierence is seen at the conserved region of Est3 surface, the TEL patch, which in hTPP1
and ScEst3 is involved in the recruitment of telomerase subunits to the telomere through the direct interac-
tion with the N-terminal domain of TERT42,45,46. e TEL regions of hTPP1 and ScEst3 surfaces are negatively
charged, while the corresponding part of HpEst3 is positively charged (Fig.7). Additionally, the second region
at the N-terminus of human TPP1 (so called NOB region, residues 91–95 of hTPP1) was shown to mediate
both telomerase recruitment to telomeres and repeat addition processivity59. is eect was sequence-specic:
replacing the NOB region of human TPP1 with NOB of its close homolog, mouse TPP1, reduced processivity of
human telomerase. In Est3 proteins, the corresponding region shows some limited sequence conservation within
yeast, with the exception of HpEst3 and some other species, but it has no similarity with animal TPP1 proteins.
ese considerations may reect dierences in intermolecular interactions in which these proteins are involved.
NMR experiments showed that free HpEst3 protein does not interact specically with oligonucleotides mod-
eling possible DNA and/or RNA environment of Est3 in telomerase complex. It is likely that the additional bind-
ing partners are required to trigger structural changes capable to open the ligand-binding interface.
In S. cerevisiae, telomerase stimulation by Est3 was linked to its ability to bind the TEN-domain of the cata-
lytic subunit39. Direct interaction between isolated recombinant Est3 and the TEN-domain was also reported
for proteins from yeasts Candida parapsilosis and Lodderomyces elongisporus60. Having well-behaved recombi-
nant HpEst3 and HpTEN, we tested the possibility of the direct interaction between these proteins by NMR.
However, chemical shi changes were not observed neither in the case of titration of 15N-labelled Est3 by TEN,
nor 15N-labelled TEN by Est3. ese results suggest that either HpEst3 lacks the ability to bind HpTEN or such
an interaction becomes possible only within the assembled H. polymorpha telomerase complex. e apparent
absence of invitro interaction between Est3 and TEN has also been reported by Tucey and Lundblad35, studying
the proteins from S. castellii species. Tucey and Lundblad contested the idea that the Est3 binds to an isolated
TEN even in S. cerevisiae. ey showed that a stable complex between ScEst3 and ScTERT does not form unless
ScTERT is pre-bound to telomerase RNA35.
Figure8. Binding interfaces of the telomeric OB fold proteins: (a) S. pombe POT1 with single-stranded
telomeric DNA fragment (magenta); (b) N-terminal OB fold domain of S.nova TEBPα complexed with TEBPβ
and a fragment of ssDNA (magenta); (c) human POT1 complexed with a peptide fragment of TPP1 (orange).
e loop 45 is shown in gold. Side chains of the ligand-interacting residues of OB fold proteins are shown in
sticks. Figure was made using UCSF Chimera v. 1.12 (https ://www.cgl.ucsf.edu/chime ra).
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However, in S. cerevisiae, Est3 does not appear to contact telomerase RNA directly—interaction with TERT/
TEN has been implicated in recruiting ScEst3 to the telomerase complex invivo24,35,3739. is assumption has
not been tested in any other yeast species. HpEst3, similar to ScEst3, was found to be unable to bind nucleic
acids. erefore, its association with HpTER is most likely to be mediated by protein–protein interactions. We
analyzed the amount of TER co-precipitated with Est3-HA from TERT and ∆tert backgrounds; however, we did
not nd any signicant reduction of the Est3-bound TER upon TERT removal. is result is consistent with the
lack of Est3—TEN binding invitro, and suggests that TERT/TEN may not serve as a factor in recruiting Est3
to TER in H. polymorpha.
Association of Est3 with TER in S. cerevisiae has been shown to be stimulated by the Est1 protein3436. Similar
Est1-dependency of the Est3-TER complex formation is described for C.albicans40. Interestingly, CaEst1 is unable
to form a stable complex with CaTER in the absence of CaEst3 invivo40. In S. cerevisiae, telomerase RNA levels
immunoprecipitated with Est1 are also diminished in est3-null mutants24,34,61, although to a lesser extent. We
found that this interdependence of Est3 and Est1 for productive TER binding can be observed in H. polymor-
pha cells as well (Fig.6). us, Est1 and Est3 are linked (at least functionally), and this connection is conserved
among budding yeast species, perhaps, even more conserved than Est3-TEN interaction. Interestingly, yeasts
C. parapsilosis and L. elongisporus lack the EST1 gene, and their Est3 proteins contain large N- and C-terminal
extensions, which are important for the interaction with TEN domain invitro60. One might speculate that these
extensions evolved to strengthen the Est3-TEN bond, thereby compensating for the Est1 loss62. us, it is pos-
sible that Est3-TEN and Est3-Est1 interactions are both able to provide a robust recruitment mechanism for
Est3, however dierent yeast species evolved to rely more on one particular interaction (like H. polymorpha, or
C. parapsilosis and L. elongisporus), or to utilize both of them (like in case of S. cerevisiae).
We uncovered a substantial reduction of tagged TERT in H. polymorpha cells lacking either Est1 or Est3. At
least in ∆est3 cells, this reduction is at the protein level, suggesting that either TERT mRNA translation or protein
stability are aected. Est3 (as well as Est1) has not been implicated in translation regulation; therefore, its eect
on the TERT stability is more plausible. Based on the results of this study, we propose that the combined action
of both Est1 and Est3 promotes productive association of TERT with telomerase RNA in H. polymorpha. Fol-
lowing the loss of any partner of a pair Est1/Est3, the second partner can no longer form a stable complex with
TER. In the absence of the Est1/Est3 pair, TERT-TER interaction weakens, leading to the increase of free TERT,
which is presumably more susceptible to degradation by cellular proteases than within a complex containing
TER. Consistent with this idea, HpTER deletion also dramatically diminishes the cellular pool of TERT-HA.
In S. cerevisiae, it has been shown that stable association of Est1 with TER invivo requires the presence of
the Pop1/Pop6/Pop7 proteins pre-bound near the Est1 RNA-binding site22,23. e regions of TER binding Est1
and the Pop proteins appear to be present in other yeast species, including H. polymoprha22,49. It is possible that
HpEst3 binds HpEst1 and the homologues of the Pop proteins in H. polymorpha, which could explain the posi-
tive eect of Est3 on Est1-TER binding observed in our experiments.
Telomerase is an evolutionarily variable enzyme. An active invivo telomerase complex includes a large
number of protein components63. eir number and structure is very dierent for various organisms. Only two
components (TER and TERT) are indispensable, although their structure is variable. Other accessory proteins
are diverse in both structure and function. For instance, in contrast to yeast Est3, the human analogue hTPP1 has
two additional domains to the C-terminus of the OB-domain: the POT1-binding domain and the TIN2-binding
domain64. POT1 protein interacts directly with the single-stranded telomeric DNA and maintains the integrity of
telomeres65. hTPP1 alone doesn’t bind ssDNA, but the complex of POT1 with hTPP1 exhibits a tenfold increase in
the anity toward ssDNA44,57. Analysis of available structures of yeast Est1 from Kluyveromyces lactis (KlEst1)66
and human Est1 homologues SMG5, SMG6, and SMG767,68 revealed high structural similarity of the Est1 TPR
domain with a family of 14-3-3 proteins. e latter are known to act as hub-proteins and mediate numerous
protein–protein interactions via binding to the unstructured fragments of their partners69. It is worth noting that
HpEst3 also has a long disordered tail, which can be involved in the binding to HpEst1 in a 14-3-3-like fashion.
Conclusions
e high-resolution structure of HpEst3 in solution has been determined. is is a rst structure of the Est3
protein determined with a large number of experimental restraints. e dynamic properties of HpEst3 were
studied, indicating that the protein has a conformational mobility of its core, typical for proteins involved in
protein–RNA and/or protein–protein interactions. NMR titration experiments indicate that free Est3 does not
interact specically with either the N-terminal domain of TERT or with DNA and/or RNA fragments mimick-
ing the probable telomerase environment. It was found, however, that both Est3 and Est1 are essential for the
formation of stable and functionally active telomerase in Hansenula polymorpha.
Materials and methods
Yeast strains. Strains used in this study are listed in Supplementary TableS2. Oligonucleotides used for
PCR during strain construction are listed in Supplementary TableS3. e DL1-L strain70 was used as a wild type
(no tag) control in all experiments. Gene replacements were performed by transformation of the DLdaduA71
or another appropriate strain with DNA integration cassettes according to a standard protocol, modied as
described72. For EST3 gene knock-out, EST3 ORF with anking regions (PCR product#1) was cloned in pUC19;
NheI/XhoI fragment of the resulting plasmid was then substituted with the SalI/XbaI fragment of the pCHLX
vector73. TERT and EST1 gene knock-outs were performed as described previously48,49. For C-terminal HA-
tagging we used a pFA6a-3HA-HpURA3 plasmid74. PCR products #2 and #3 were cloned at the SalI/XmaI and
PmeI/ClaI sites (respectively) of the pFA6a-3HA-HpURA3 vector for construction of the TERT-HA strain; PCR
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products #4 and #5—for the EST3-HA strain; PCR products #6 and #7—for the EST1-HA strain. Correct inte-
gration of the cassettes and gene replacements were veried by PCR.
Spotassay. Several ∆est3 colonies aer transformation were grown overnight (“passage 1”) in 2ml of com-
plete minimal medium75 without leucine (SC-LEU); 1ml of culture was used for genotype verication. e rest
was inoculated in 100ml of SC-LEU (OD600 ~ 0.05) and grown overnight at 37°C (“passage 2”). Two independ-
ent ∆est3 transformants were passaged six more times. Each time, 10μl aliquots of cultures with OD600 ~ 0.05
(along with three tenfold dilutions) were spotted onto YPD (1% yeast extract, 2% peptone, 2% glucose) agar plate
and grown for 2days at 37°C. 50ml of the overnight cultures during each passage were collected and used for
telomere length measurements.
Telomere Southern blots. PstI-digested genomic DNA was separated on 1% agarose gel, then transferred
to a nylon membrane (Whatman Nytran SuPerCharge). Southern hybridization was performed according to the
standard protocol 76. 5-radiolabelled C4 oligonucleotide (5-(CGC CAC CC)4-3) was used as a probe.
Immunoprecipitation(IP)experiments. Typically, yeast cells were grown to OD600 ~ 1 in 400ml of YPD
(1% yeast extract, 2% peptone, 2% glucose) medium at 37°C. S In case of telomerase null cells, we used only
freshly prepared knock-outs for IP experiments. Several colonies aer transformation were grown overnight in
2ml of SC-LEU: 1ml of culture was used for genotype verication, and to another 1ml portion we added 2ml
of YPD and grew overnight. en, these cultures were diluted in 400ml of YPD and grown to OD600 ~ 1.
Cells were collected by centrifugation, resuspended in 1ml of binding buer [20mM HEPES-NaOH pH
7.5, 2mM MgCl2, 10% glycerol, 0.1% Nonidet P-40, 150mM NaCl, 1mM DTT, Halt Protease and Phosphatase
Inhibitor Cocktail (ermo Scientic)] and broken by glass beads in Precellys Evolution homogenizer. Cell
extracts were cleared by centrifugation (16,000g, 20min). 1ml of lysates were incubated with 40μl (1:1 suspen-
sion) of anti-HA agarose (Roche, A2095) at + 4°C for 1h. Beads were washed three times with binding buer.
Half of the beads were analyzed by Western blot and the other half by qRT-PCR.
For experiments shown in Fig.2c, d, 1ml of TERT-HA EST3 lysate and 4ml of TERT-HA ∆est3 lysate (pre-
pared from 400ml and 1,600ml of YPD cultures, respectively) were incubated with 40μl of anti-HA beads. For
Western blot we used 1/10 (TERT-HA EST3) and 1/2 (TERT-HA ∆est3) of beads; for telomerase activity assay—
1/20 (TERT-HA EST3) and 1/4 (TERT-HA ∆est3) of beads. All IP experiments were performed in triplicate.
Westernblotanalysis. Proteins were eluted from anti-HA resin by incubation with 15μl of HU buer
(7M urea, 5% SDS, 0.2M Tris–HCl pH 6.8, 1mM EDTA, 0.2% bromophenol blue) at 95°C for 10min. Eluates
or input samples were separated by 6% (TERT-HA and Est1-HA) or 12% (Est3-HA) SDS-PAGE. Anti-HA-HRP
antibodies (clone 3F10, Sigma-Aldrich) at 1:2000 and a subsequent Western Bright ECL Kit (Advansta) were
used for detection. e linearity of the TERT-HA signal was veried by analyzing serial two-fold dilutions of IP
samples and extracts containing TERT-HA.
qRT-PCR. ½ portions of anti-HA beads or 3μl aliquots of extracts (0.3% of input) were diluted in a binding
buer to a nal volume 100μl, supplemented with SDS (0.1% nal); then treated with 40μg of proteinase K at
37°C for 30min. RNA was extracted with phenol/chloroform, precipitated with EtOH and dissolved in 20μl of
mQ water. Samples were treated with DNase I (ermo Fisher Scientic). cDNA synthesis and PCR reactions
were performed as described49, with the only modication being that we used Maxima RT (ermo Fisher
Scientic) for cDNA production. Data was represented as yields (% of input), the values calculated using the
formula: 2^(Ct(input)−Ct(beads))×2×0.3.
Telomerase activity assay. 50 μl mixtures [containing 15μl anti-HA beads (resuspended in binding
buer without Nonidet P-40), 1μM HD5 oligonucleotide (5-AAA AAG GGT GGC G-3), 50mM Tris–HCl pH8,
1mM spermidine, 1mM DTT, dATP/dTTP/dCTP (50μM each), and 3.75μM α-32P-dGTP (800Ci/mmol)]
were incubated at 30°C for 30min. Reactions were stopped by proteinase K treatment, extracted with phenol/
chloroform and precipitated with EtOH. 5-32P-labeled oligonucleotide 5-AAA AAA GGG TGG C-3 was added
aer proteinase K treatment served as a loading control. Products were resolved on 10% denaturing PAGE.
ExpressionofHpEst3and HpTEN. HpEst3 and HpTEN were expressed and puried as described in
Refs.47,77,78 correspondingly.
RNAandDNAsynthesis. DNA and RNA oligonucleotides were assembled in an MM-12 synthesizer (Bio-
automation) with the phosphoramidite method, according to the manufacturer’s recommendations at 25µmol
scale. Synthetic procedure is described in detail earlier78.
NMRspectroscopy. e NMR samples in concentration of 0.4mM for 13C,15N-labeled HpEst3 and 0.2–
0.4mM for 15N-labeled protein were prepared in 90% H2O/10% D2O, 100mM KCl, and 20mM potassium
phosphate buer (pH 6.5). DTT in concentration of 3mM was added to the nal solution to prevent oxidation
of three cysteine residues (C29, C61 and C63). Triple-resonance (1H,13C,15N) spectra were acquired at 298K
on a Bruker Avance III HD 700MHz spectrometer equipped with a quadruple resonance (1H,13C,15N,31P) QCI
CryoProbe. 15N-1H HSQC and SOFAST HMQC spectra on 15N-labeled HpEst3 in NMR titration experiments
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were acquired at 298K on a Bruker Avance 600MHz spectrometer equipped with a triple resonance (1H,13C,15N)
TXI probe. All spectra were processed by NMRPipe79, and analyzed using NMRFAM-SPARKY80.
NMR structure determination. e family of 20 NMR structures of HpEst3 was calculated using con-
formational restraints for 2,262 inter-proton distances and 228 backbone dihedral angles (Table1). A set of
dihedral angles was obtained from the analysis of the 1HN, 1Hα, 15N, 13Cα, 13Cβ and 13C chemical shis using the
TALOS + soware 81 for the residues located in the well-ordered regions of the protein core, as dened by NMR
relaxation experiments and the RCI (Random Coil Index) approach82. NOEs, used as distance restraints in struc-
ture calculation, were obtained from the analysis of cross-peak intensities in the 3D 13C-1H and 15N-1H HSQC-
NOESY spectra. e intra-residue and sequential cross-peaks of NOESY spectra were assigned manually, while
the rest of the cross-peaks were assigned using the automatic iterative procedure of spectra assignment/struc-
ture calculation implemented in ARIA 2.3 soware83. e automatic assignment and the inter-proton distances
provided at the last iteration of the ARIA 2.3 protocol were further manually veried by multiple steps of the
structure renement accomplished using the simulated annealing protocol of the CNS 1.21 soware package84.
Structure renement included a high-temperature torsion-angle molecular dynamics stage followed by a slow-
cooling torsion-angle phase, a second slow-cooling phase in Cartesian space and Powell energy minimization.
Database values of conformational torsion angle pseudopotentials85 were implemented during the nal cycles of
the calculations to improve the quality of protein backbone conformation. Structure renement was performed
until no NOE violations larger than 0.5Å and no dihedral angle violations higher than 5° occurred. e restraint
violations and structure quality were assessed using the CNS tools, Procheck-NMR86, and in-house soware and
utilities. At the last iteration of the renement protocol 200 structures were calculated using 2,262 unambiguous
distance and 228 dihedral angle restraints. e nal family of 20 NMR structures was ltered out in accordance
with the lowest-energy criterion. Statistics for the determined NMR structures are presented in Table1. Struc-
ture visualization and analysis were carried out using PyMOL (Schrödinger LLC).
NMRdynamicsanalysis. R1, R2 and 1H-15N heteronuclear NOE data sets of 15N uniformly labeled HpEst3
were collected at 298K on a Bruker Avance III HD 700MHz spectrometer. e delays for the R1 relaxation rate
experiments were 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.58, 0.64, 0.8, 1.0, 1.3, 1.8, 2.5s; and for the R2 relaxation
rate experiments were 0, 17, 33.9, 50.9, 67.8, 84.8, 101.8, 118.7, 135.7, 152.6, 169.6, 186.6, 203.5, 237.4, 271.4,
305.3ms. e excitation time for 1H in the 1H-15N heteronuclear NOE experiments was 4.0s. Spectra were pro-
cessed using NMRPipe soware79. e nonlinear tting of the integrated peak volumes in the pseudo 3D spectra
of the relaxation experiments and the calculation of standard deviations were accomplished using the nlinLS
procedure. e values of R1 and R2 were then calculated from the table of relative peak intensities, produced by
NMRPipe and nlinLS, using RelaxFit, which was written in-house87. e standard deviations of the 15N-1H NOE
values were calculated using the RMS noise of the background regions88 and were further checked and corrected
using two independently collected experimental data sets. e analysis of the R1, R2 and 1H,15N-NOE values was
carried out using a model-free formalism using the RelaxFit program87. To determine the rotational diusion
tensor, all of the isotropic, axially symmetric, and fully asymmetric molecular tumbling models were tested. e
values of the correlation time of protein tumbling or the diusion tensor axis were then used to t models of
internal motions for the backbone HN vectors of the amino acid residues.
NMRtitrationexperiments. Protein–protein and protein-nucleic acids interactions were tested using the
15N,1H SOFAST HMQC experiments measured at 25°C and 600MHz 1H frequency. A single 15N-labelled pro-
tein (HpEst3 or HpTEN, concentration between 0.15 and 0.40mM) was used to monitor intermolecular inter-
actions, while one or more unlabelled substances were gradually added to the sample. Reaction mixtures were
studied in a buer which contained 100mM KCl, 20mM potassium phosphate (pH = 6.5), 0.02% NaN3, and
3mM DTT. e pH of the unlabelled protein samples was adjusted by dialysis against this buer. ese samples
were then aliquoted and freeze-dried to prevent a change in the concentration of the 15N-labeled protein during
the titration. Short unlabelled oligonucleotides (see Supplementary TableS1) were used to examine protein-NA
interactions. e pH values of the oligonucleotides were adjusted to be identical to the pH of 15N-labelled protein
solution, and then aliquoted and freeze-dried. DNA-RNA heteroduplexes were prepared using the annealing
protocol, described earlier78. e RNA hairpin was prepared at 70°C and low concentration of RNA (50μM)
to avoid oligomerisation. A RiboLock RNase inhibitor (ermo Fisher Scientic), in concentration of 700U/
ml was added to the samples containing the single-stranded RNA in order to inhibit RNA cleavage. e proto-
col for carrying out NMR titration experiments is generally identical to that previously described by us for the
HpTEN78.
Electrophoreticmobilityshiftassay(EMSA). EMSA was performed essentially as described earlier74
the following modications. 1 μM fG4 oligonucleotide (FAM-5-(GGG TGG CG)4) was incubated with an
increasing amount of Est3 (concentration range: 0, 1, 3, 10μM). Reactions were performed in a solution of
20mM potassium phosphate (pH = 6.5), 100mM KCl, and 3mM DTT. Products were separated in an 8% non-
denaturing polyacrylamide gel (19:1).
Equipment and settings for gel/blot images. Gels and blots from this study were acquired as described
earlier74 with the following modications. e Western blot images were acquired using the “Chemi Hi Resolu-
tion” application. e gels from the telomerase activity assays were acquired on the Typhoon FLA 7000 (GE
Healthcare) imaging system using the “Phosphor” method. Processing (cropping and contrast adjustments) was
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performed in ImageLab 5.2.1 soware, ImageQuant TL 7.0 or Adobe Photoshop CC 2018. Contrast adjustments
were applied equally across the entire images (including controls).
Codeavailability
e structural data and experimental restraints used in calculations have been submitted to the Protein Data
Bank with accession number 6Q44.
Received: 17 November 2019; Accepted: 19 June 2020
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Acknowledgements
Authors are grateful to the Moscow State University (Russia) for the opportunity to use the NMR facilities and
the supercomputer SKIF Lomonosov. Authors acknowledge the support from the Russian Government Program
of Competitive Growth of Kazan Federal University among the world’s leading academic centres and the NMR
equipment at the KFU Center of shared facilities. is work was supported by the Russian Science Foundation
(Grant № 19-14-00115 to V.I.P. and 18-73-00068 to A.B.M.).
Authorcontributions
O.A.P. and M.I.Z. carried out protein expression and purication; N.M.S. and A.N.M. performed telomerase
assays and experiments with yeast; S.S.M. and S.V.E. performed the NMR experiments; A.B.M. carried out NMR
structure calculations and molecular modelling; V.I.P. performed the NMR relaxation data analysis; E.V.R.,
O.A.D. and V.I.P. designed the research, analyzed data and wrote the manuscript.
Competing interests
e authors declare no competing interests.
Additionalinformation
Supplementary information is available for this paper at https ://doi.org/10.1038/s4159 8-020-68107 -x.
Correspondence and requests for materials should be addressed to O.A.D.orV.I.P.
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... In S. cerevisiae, Ku is one of the accessory subunits of telomerase stably bound to its RNA component. We found that HpTER cannot be efficiently co-purified with Ku80-HA, while it robustly binds TERT-HA, Est1-HA, and Est3-HA in the same experimental conditions ( Figure 5-figure supplement 2F, Shepelev et al., 2020), suggesting that HpKu is not a stable telomerase component. This does not exclude, however, the possibility of weak or transient association between Ku and telomerase in H. polymorpha. ...
... Southern blot experiments were carried out as previously described (Shepelev et al., 2020). TRF length were calculated using the ImageQuant TL 1D software version 7.0. ...
... RNA Co-IP experiments were performed as described in Shepelev et al., 2020. Protein expression and purification Rif1 fragments for EMSA were expressed and purified as HpRap1B protein (Malyavko et al., 2019). ...
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