scholarly journals Characterization of recombinant Saccharomyces cerevisiae telomerase core enzyme purified from yeast

2005 ◽  
Vol 390 (1) ◽  
pp. 169-176 ◽  
Author(s):  
Xin-Hua Liao ◽  
Ming-Liang Zhang ◽  
Cui-Ping Yang ◽  
Lu-Xia Xu ◽  
Jin-Qiu Zhou
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Telomerase Activity ◽  
Glutathione S Transferase ◽  
Core Complex ◽  
Core Enzyme ◽  
Ammonium Sulphate Fractionation ◽  
Telomerase Regulation

Telomerase is a cellular reverse transcriptase that elongates the single-stranded chromosome ends and oligonucleotides in vivo and in vitro. In Saccharomyces cerevisiae, Est2p (telomerase catalytic subunit) and Tlc1 (telomerase RNA template subunit) constitute the telomerase core complex. We co-overexpressed GST (glutathione S-transferase)–Est2p and Tlc1 in S. cerevisiae, and reconstituted the telomerase activity. The GST–Est2p–Tlc1 complex was partially purified by ammonium sulphate fractionation and affinity chromatography on glutathione beads, and the partially purified telomerase did not contain the other two subunits of the telomerase holoenzyme, Est1p and Est3p. The purified recombinant GST–Est2p–Tlc1 telomerase core complex could specifically add nucleotides on to the single-stranded TG1–3 primer in a processive manner, but could not translocate to synthesize more than one telomeric repeat. The purified telomerase core complex exhibited different activities when primers were paired with the Tlc1 template at different positions. The procedure of reconstitution and purification of telomerase core enzyme that we have developed now allows for further mechanistic studies of the functions of other subunits of the telomerase holoenzyme as well as other telomerase regulation proteins.

scholarly journals The Biochemical Activities of the Saccharomyces cerevisiae Pif1 Helicase Are Regulated by Its N-Terminal Domain

Genes ◽  
2019 ◽  
Vol 10 (6) ◽  
pp. 411 ◽  
Author(s):  
Nickens ◽  
Sausen ◽  
Bochman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Telomerase Activity ◽  
Full Length ◽  
Dna Unwinding ◽  
Genome Maintenance ◽  
Terminal Domain ◽  
Pif1 Helicase ◽  
Telomerase Regulation

: Pif1 family helicases represent a highly conserved class of enzymes involved in multiple aspects of genome maintenance. Many Pif1 helicases are multi-domain proteins, but the functions of their non-helicase domains are poorly understood. Here, we characterized how the N-terminal domain (NTD) of the Saccharomyces cerevisiae Pif1 helicase affects its functions both in vivo and in vitro. Removal of the Pif1 NTD alleviated the toxicity associated with Pif1 overexpression in yeast. Biochemically, the N-terminally truncated Pif1 (Pif1ΔN) retained in vitro DNA binding, DNA unwinding, and telomerase regulation activities, but these activities differed markedly from those displayed by full-length recombinant Pif1. However, Pif1ΔN was still able to synergize with the Hrq1 helicase to inhibit telomerase activity in vitro, similar to full-length Pif1. These data impact our understanding of Pif1 helicase evolution and the roles of these enzymes in the maintenance of genome integrity.

scholarly journals The biochemical activities of the Saccharomyces cerevisiae Pif1 helicase are regulated by its N-terminal domain

2019 ◽  
Author(s):  
David G. Nickens ◽  
Christopher W. Sausen ◽  
Matthew L. Bochman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Telomerase Activity ◽  
Full Length ◽  
Genome Maintenance ◽  
Over Expression ◽  
Terminal Domain ◽  
Pif1 Helicase ◽  
Telomerase Regulation

AbstractPIF1 family helicases represent a highly conserved class of enzymes involved in multiple aspects of genome maintenance. Many PIF1 helicase are multi-domain proteins, but the functions of their non-helicase domains are poorly understood. Here, we characterized how the N-terminal domain (NTD) of theSaccharomyces cerevisiaePif1 helicase affects its functions bothin vivoandin vitro. Removal of the Pif1 NTD alleviated the toxicity associated with Pif1 over-expression in yeast. Biochemically, the N-terminally truncated Pif1 (Pif1ΔN) retainedin vitroDNA binding, DNA unwinding, and telomerase regulation activities, but these activities differed markedly from those displayed by full-length recombinant Pif1. However, Pif1ΔN was still able to synergize with the Hrq1 helicase to inhibit telomerase activityin vitro, similar to full-length Pif1. These data impact our understanding of PIF1 helicase evolution and the roles of these enzymes in the maintenance of genome integrity.

scholarly journals Expression and Characterization of the Flocculin Flo11/Muc1, a Saccharomyces cerevisiae Mannoprotein with Homotypic Properties of Adhesion

2007 ◽  
Vol 6 (12) ◽  
pp. 2214-2221 ◽  
Author(s):  
Lois M. Douglas ◽  
Li Li ◽  
Yang Yang ◽  
A. M. Dranginis
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Biofilm Formation ◽  
In Vitro System ◽  
Glycosylphosphatidylinositol Anchor ◽  
Fungal Adhesion ◽  
Very High ◽  
Molecular Weight Form

ABSTRACT The Flo11/Muc1 flocculin has diverse phenotypic effects. Saccharomyces cerevisiae cells of strain background Σ1278b require Flo11p to form pseudohyphae, invade agar, adhere to plastic, and develop biofilms, but they do not flocculate. We show that S. cerevisiae var. diastaticus strains, on the other hand, exhibit Flo11-dependent flocculation and biofilm formation but do not invade agar or form pseudohyphae. In order to study the nature of the Flo11p proteins produced by these two types of strains, we examined secreted Flo11p, encoded by a plasmid-borne gene, in which the glycosylphosphatidylinositol anchor sequences had been replaced by a histidine tag. A protein of approximately 196 kDa was secreted from both strains, which upon purification and concentration, aggregated into a form with a very high molecular mass. When secreted Flo11p was covalently attached to microscopic beads, it conferred the ability to specifically bind to S. cerevisiae var. diastaticus cells, which flocculate, but not to Σ1278b cells, which do not flocculate. This was true for the 196-kDa form as well as the high-molecular-weight form of Flo11p, regardless of the strain source. The coated beads bound to S. cerevisiae var. diastaticus cells expressing FLO11 and failed to bind to cells with a deletion of FLO11, demonstrating a homotypic adhesive mechanism. Flo11p was shown to be a mannoprotein. Bead-to-cell adhesion was inhibited by mannose, which also inhibits Flo11-dependent flocculation in vivo, further suggesting that this in vitro system is a useful model for the study of fungal adhesion.

scholarly journals Yeast Est2p Affects Telomere Length by Influencing Association of Rap1p with Telomeric Chromatin

2008 ◽  
Vol 28 (7) ◽  
pp. 2380-2390 ◽  
Author(s):  
Hong Ji ◽  
Christopher J. Adkins ◽  
Bethany R. Cartwright ◽  
Katherine L. Friedman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Telomere Length ◽  
Specific Binding ◽  
Telomerase Activity ◽  
Negative Regulator ◽  
Wild Type ◽  
Telomeric Dna ◽  
Telomere Length Homeostasis

ABSTRACT In Saccharomyces cerevisiae, the sequence-specific binding of the negative regulator Rap1p provides a mechanism to measure telomere length: as the telomere length increases, the binding of additional Rap1p inhibits telomerase activity in cis. We provide evidence that the association of Rap1p with telomeric DNA in vivo occurs in part by sequence-independent mechanisms. Specific mutations in EST2 (est2-LT) reduce the association of Rap1p with telomeric DNA in vivo. As a result, telomeres are abnormally long yet bind an amount of Rap1p equivalent to that observed at wild-type telomeres. This behavior contrasts with that of a second mutation in EST2 (est2-up34) that increases bound Rap1p as expected for a strain with long telomeres. Telomere sequences are subtly altered in est2-LT strains, but similar changes in est2-up34 telomeres suggest that sequence abnormalities are a consequence, not a cause, of overelongation. Indeed, est2-LT telomeres bind Rap1p indistinguishably from the wild type in vitro. Taken together, these results suggest that Est2p can directly or indirectly influence the binding of Rap1p to telomeric DNA, implicating telomerase in roles both upstream and downstream of Rap1p in telomere length homeostasis.

scholarly journals Establishment and Characterization of a Reliable Xenograft Model of Hodgkin Lymphoma Suitable for the Study of Tumor Origin and the Design of New Therapies

Cancers ◽  
2018 ◽  
Vol 10 (11) ◽  
pp. 414 ◽  
Author(s):  
Radhia M’kacher ◽  
Monika Frenzel ◽  
Mustafa Al Jawhari ◽  
Steffen Junker ◽  
Corina Cuceu ◽  
...  
Keyword(s):  
Animal Model ◽  
Histone Deacetylase ◽  
High Frequency ◽  
Histone Deacetylase Inhibitor ◽  
Telomerase Activity ◽  
Nsg Mice ◽  
Deacetylase Inhibitor

To identify the cells responsible for the initiation and maintenance of Hodgkin lymphoma (HL) cells, we have characterized a subpopulation of HL cells grown in vitro and in vivo with the aim of establishing a reliable and robust animal model for HL. To validate our model, we challenged the tumor cells in vivo by injecting the alkylating histone-deacetylase inhibitor, EDO-S101, a salvage regimen for HL patients, into xenografted mice. Methodology: Blood lymphocytes from 50 HL patients and seven HL cell lines were used. Immunohistochemistry, flow cytometry, and cytogenetics analyses were performed. The in vitro and in vivo effects of EDO-S101 were assessed. Results: We have successfully determined conditions for in vitro amplification and characterization of the HL L428-c subline, containing a higher proportion of CD30−/CD15− cells than the parental L428 cell line. This subline displayed excellent clonogenic potential and reliable reproducibility upon xenografting into immunodeficient NOD-SCID-gamma (−/−)(NSG) mice. Using cell sorting, we demonstrate that CD30−/CD15− subpopulations can gain the phenotype of the L428-c cell line in vitro. Moreover, the human cells recovered from the seventh week after injection of L428-c cells into NSG mice were small cells characterized by a high frequency of CD30−/CD15− cells. Cytogenetic analysis demonstrated that they were diploid and showed high telomere instability and telomerase activity. Accordingly, chromosomal instability emerged, as shown by the formation of dicentric chromosomes, ring chromosomes, and breakage/fusion/bridge cycles. Similarly, high telomerase activity and telomere instability were detected in circulating lymphocytes from HL patients. The beneficial effect of the histone-deacetylase inhibitor EDO-S101 as an anti-tumor drug validated our animal model. Conclusion: Our HL animal model requires only 103 cells and is characterized by a high survival/toxicity ratio and high reproducibility. Moreover, the cells that engraft in mice are characterized by a high frequency of small CD30−/CD15− cells exhibiting high telomerase activity and telomere dysfunction.

scholarly journals Functional characterization of Ost3p. Loss of the 34-kD subunit of the Saccharomyces cerevisiae oligosaccharyltransferase results in biased underglycosylation of acceptor substrates.

1995 ◽  
Vol 130 (3) ◽  
pp. 567-577 ◽  
Author(s):  
D Karaoglu ◽  
D J Kelleher ◽  
R Gilmore
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Functional Characterization ◽  
Null Mutant ◽  
Wild Type ◽  
Membrane Glycoproteins ◽  
En Bloc ◽  
Oligosaccharide Moiety

Within the lumen of the rough endoplasmic reticulum, oligosaccharyltransferase catalyzes the en bloc transfer of a high mannose oligosaccharide moiety from the lipid-linked oligosaccharide donor to asparagine acceptor sites in nascent polypeptides. The Saccharomyces cerevisiae oligosaccharyltransferase was purified as a heteroligomeric complex consisting of six subunits (alpha-zeta) having apparent molecular masses of 64 kD (Ost1p), 45 kD (Wbp1p), 34 kD, 30 kD (Swp1p), 16 kD, and 9 kD. Here we report a structural and functional characterization of Ost3p which corresponds to the 34-kD gamma-subunit of the oligosaccharyltransferase. Unlike Ost1p, Wbp1p, and Swp1p, expression of Ost3p is not essential for viability of yeast. Instead, ost3 null mutant yeast grow at wild-type rates on solid or in liquid media irrespective of culture temperature. Nonetheless, detergent extracts prepared from ost3 null mutant membranes are twofold less active than extracts prepared from wild-type membranes in an in vitro oligosaccharyltransferase assay. Furthermore, loss of Ost3p is accompanied by significant underglycosylation of soluble and membrane-bound glycoproteins in vivo. Compared to the previously characterized ost1-1 mutant in the oligosaccharyltransferase, and the alg5 mutant in the oligosaccharide assembly pathway, ost3 null mutant yeast appear to be selectively impaired in the glycosylation of several membrane glycoproteins. The latter observation suggests that Ost3p may enhance oligosaccharide transfer in vivo to a subset of acceptor substrates.

scholarly journals Characterization of Interactions between PinX1 and Human Telomerase Subunits hTERT and hTR

2004 ◽  
Vol 279 (50) ◽  
pp. 51745-51748 ◽  
Author(s):  
Soma S. R. Banik ◽  
Christopher M. Counter
Keyword(s):  
Telomerase Activity ◽  
Protein Subunit ◽  
Human Telomerase Reverse Transcriptase ◽  
Telomere Elongation ◽  
Cellular Context ◽  
Core Components ◽  
Human Telomerase

The addition of telomeric repeats to chromosome ends by the enzyme telomerase is a highly orchestrated process. Although much is known regarding telomerase catalytic activityin vitro, less is known about how this activity is regulatedin vivoto ensure proper telomere elongation. One protein that appears to be involved in negatively regulating telomerase functionin vivois PinX1 because overexpression of PinX1 inhibits telomerase activity and causes telomere shortening. To understand the nature of this repression, we characterized the interactions among PinX1 and the core components of telomerase, the human telomerase reverse transcriptase (hTERT) and associated human telomerase RNA (hTR). We now show thatin vitroPinX1 binds directly to the hTERT protein subunit, primarily to the hTR-binding domain, as well as to the hTR subunit. However, in a cellular context, the association of PinX1 with hTR is dependent on the presence of hTERT. Taken together, we suggest that PinX1 represses telomerase activityin vivoby binding to the assembled hTERT·hTR complex.

scholarly journals Telomerase and Pluripotency Factors Jointly Regulate Stemness in Pancreatic Cancer Stem Cells

Cancers ◽  
2021 ◽  
Vol 13 (13) ◽  
pp. 3145
Author(s):  
Karolin Walter ◽  
Eva Rodriguez-Aznar ◽  
Monica S. Ventura Ferreira ◽  
Pierre-Olivier Frappart ◽  
Tabea Dittrich ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Tumor Biology ◽  
Telomerase Activity ◽  
Pancreatic Cancer Cells ◽  
Sphere Formation ◽  
Telomerase Regulation ◽  
Pancreatic Cancer Stem Cells

To assess the role of telomerase activity and telomere length in pancreatic CSCs we used different CSC enrichment methods (CD133, ALDH, sphere formation) in primary patient-derived pancreatic cancer cells. We show that CSCs have higher telomerase activity and longer telomeres than bulk tumor cells. Inhibition of telomerase activity, using genetic knockdown or pharmacological inhibitor (BIBR1532), resulted in CSC marker depletion, abrogation of sphere formation in vitro and reduced tumorigenicity in vivo. Furthermore, we identify a positive feedback loop between stemness factors (NANOG, OCT3/4, SOX2, KLF4) and telomerase, which is essential for the self-renewal of CSCs. Disruption of the balance between telomerase activity and stemness factors eliminates CSCs via induction of DNA damage and apoptosis in primary patient-derived pancreatic cancer samples, opening future perspectives to avoid CSC-driven tumor relapse. In the present study, we demonstrate that telomerase regulation is critical for the “stemness” maintenance in pancreatic CSCs and examine the effects of telomerase inhibition as a potential treatment option of pancreatic cancer. This may significantly promote our understanding of PDAC tumor biology and may result in improved treatment for pancreatic cancer patients.

scholarly journals A feedback-loop between telomerase activity and stemness factors regulates PDAC stem cells

2020 ◽  
Author(s):  
Karolin Walter ◽  
Eva Rodriguez-Aznar ◽  
Monica S. Ventura Ferreira ◽  
Pierre-Olivier Frappart ◽  
Tabea Dittrich ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Cell Biology ◽  
Feedback Loop ◽  
Telomerase Activity ◽  
Ductal Adenocarcinoma ◽  
Pancreatic Cancer Cells ◽  
Telomerase Regulation

AbstractTo date, it is still unclear how cancer stem cells (CSCs) regulate their stemness properties, and to what extent they share common features with normal stem cells. Telomerase regulation is a key factor in stem cell maintenance. In this study, we investigate how telomerase regulation affects cancer stem cell biology in pancreatic ductal adenocarcinoma (PDAC), and delineate the mechanisms by which telomerase activity and CSC properties are linked. Using primary patient-derived pancreatic cancer cells, we show that CSCs have higher telomerase activity and longer telomeres than bulk tumor cells. Inhibition of telomerase activity, using genetic TERT-knockdown or pharmacological inhibitor (BIBR1532) resulted in CSC marker depletion in vitro, and reduced tumorigenicity in vivo. Furthermore, we identify a positive feedback loop between stemness factors (KLF4, SOX2, OCT3/4, NANOG) and telomerase, which is essential for the self-renewal of pancreatic CSCs. Disruption the balance between telomerase activity and stemness factors, eliminates CSCs via induction of DNA damage and apoptosis, opening future perspectives to avoid CSC driven therapy resistance and tumor relapse in PDAC patients.

scholarly journals The Saccharomyces cerevisiae Hrq1 and Pif1 DNA helicases synergistically modulate telomerase activity in vitro

2018 ◽  
Author(s):  
David G. Nickens ◽  
Cody M. Rogers ◽  
Matthew L. Bochman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Telomere Length ◽  
Telomerase Activity ◽  
Dna Helicases ◽  
Catalytic Inhibitor ◽  
Catalytically Active ◽  
High Concentrations ◽  
Telomere Length Homeostasis

ABSTRACTTelomere length homeostasis is vital to maintaining genomic stability and is regulated by multiple factors, including telomerase activity and DNA helicases. The Saccharomyces cerevisiae Pif1 helicase was the first discovered catalytic inhibitor of telomerase, but recent experimental evidence suggests that Hrq1, the yeast homolog of the disease-linked human RecQ-like helicase 4 (RECQL4), plays a similar role via an undefined mechanism. Using yeast extracts enriched for telomerase activity and an in vitro primer extension assay, here we determined the effects of recombinant wild-type and inactive Hrq1 and Pif1 on total telomerase activity and telomerase processivity. We found that titrations of these helicases alone have equal-but-opposite biphasic effects on telomerase, with Hrq1 stimulating activity at high concentrations. When the helicases were combined in reactions, however, they synergistically inhibited or stimulated telomerase activity depending on which helicase was catalytically active. These results suggest that Hrq1 and Pif1 interact and that their concerted activities ensure proper telomere length homeostasis in vivo. We propose a model in which Hrq1 and Pif1 cooperatively contribute to telomere length homeostasis in yeast.

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