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The Hsp82 molecular chaperone promotes a switch between unextendable and extendable telomere states.

DeZwaan DC, Toogun OA, Echtenkamp FJ, Freeman BC - Nat. Struct. Mol. Biol. (2009)

Bottom Line: We have established an in vitro yeast telomere system in which Stn1-Ten1-unextendable or telomerase-extendable states can be observed.Both assemblies are Cdc13 dependent, as the Cdc13 C-terminal region supports Stn1-Ten1 interactions and the N-terminal region contains a telomerase-activation function.Notably, the yeast Hsp90 chaperone Hsp82 mediates the switch between the telomere capping and extending structures by modulating the DNA binding activity of Cdc13.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Developmental Biology, University of Illinois, Urbana-Champaign, Urbana, Illinois, USA.

ABSTRACT
Distinct protein assemblies are nucleated at telomeric DNA to both guard the ends from damage and lengthen the DNA after replication. In yeast, Cdc13 recruits either Stn1-Ten1 to form a protective cap or the telomerase holoenzyme to extend the DNA. We have established an in vitro yeast telomere system in which Stn1-Ten1-unextendable or telomerase-extendable states can be observed. Both assemblies are Cdc13 dependent, as the Cdc13 C-terminal region supports Stn1-Ten1 interactions and the N-terminal region contains a telomerase-activation function. Notably, the yeast Hsp90 chaperone Hsp82 mediates the switch between the telomere capping and extending structures by modulating the DNA binding activity of Cdc13. Taken together, our data show that the Hsp82 chaperone facilitates telomere DNA maintenance by promoting transitions between two operative complexes and by reducing the potential for binding events that would otherwise block the assembly of downstream structures.

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Stn1 and Ten1 cooperate with Cdc13 to form an unextendable telomere protein-DNA complex in vitro. (a) The effect of an Stn1/Ten1 protein titration (50, 100, 250, 500 and 1000 nM) on telomerase DNA extension in the presence or absence of Cdc13 (250 nM) was determined using 23- or 7-base 3′-overhang DNAs. For comparison, the activity of unsupplemented and Cdc13 supplemented telomerase extract is shown. (b) Cdc13-2 does not form an unextendable DNA complex with Stn1/Ten1. Telomerase DNA extension reactions were performed using a 23-nucleotide 3′-overhang substrate and the reactions were supplemented with Cdc13 (FL) (250 nM) or Cdc13-2 (2) (250 nM) with or without Stn1/Ten1 (250 nM), as marked. All extension reactions were supplemented with a loading control primer (arrow) prior to precipitation and electrophoretic resolution and the +1 position for each DNA substrates is indicated. (c) The carboxyl-terminus of Cdc13 harbors an Stn1/Ten1 interaction surface. Fluorescence anisotropy with a fluorescein-labeled 15-base 3′-overhang telomeric oligonucleotide was used to detect DNA association; the 15-base DNA section provides a single Cdc13 binding site. The anisotropy was measured for each Cdc13 derivative (100 nM) alone and in the presence of Stn1/Ten1 (100 nM). The quantified data represent average values (mean +/− s.d.) from 5 independent assays.
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Figure 3: Stn1 and Ten1 cooperate with Cdc13 to form an unextendable telomere protein-DNA complex in vitro. (a) The effect of an Stn1/Ten1 protein titration (50, 100, 250, 500 and 1000 nM) on telomerase DNA extension in the presence or absence of Cdc13 (250 nM) was determined using 23- or 7-base 3′-overhang DNAs. For comparison, the activity of unsupplemented and Cdc13 supplemented telomerase extract is shown. (b) Cdc13-2 does not form an unextendable DNA complex with Stn1/Ten1. Telomerase DNA extension reactions were performed using a 23-nucleotide 3′-overhang substrate and the reactions were supplemented with Cdc13 (FL) (250 nM) or Cdc13-2 (2) (250 nM) with or without Stn1/Ten1 (250 nM), as marked. All extension reactions were supplemented with a loading control primer (arrow) prior to precipitation and electrophoretic resolution and the +1 position for each DNA substrates is indicated. (c) The carboxyl-terminus of Cdc13 harbors an Stn1/Ten1 interaction surface. Fluorescence anisotropy with a fluorescein-labeled 15-base 3′-overhang telomeric oligonucleotide was used to detect DNA association; the 15-base DNA section provides a single Cdc13 binding site. The anisotropy was measured for each Cdc13 derivative (100 nM) alone and in the presence of Stn1/Ten1 (100 nM). The quantified data represent average values (mean +/− s.d.) from 5 independent assays.

Mentions: In addition to Cdc13, Stn1 and Ten1 serve in telomere capping in vivo1. To determine whether FL-Cdc13 can function with Stn1 and Ten1 we purified the proteins (Supplementary Fig. 2a) and tested the effects of the proteins on telomerase activity. In the presence of Cdc13 we observed a striking reduction in DNA extension with increasing Stn1/Ten1 levels using the 23-base 3′-overhang DNA but not the 7-base substrate (Fig. 3a and Supplementary Fig. 2b). In the absence of Cdc13, Stn1/Ten1 had no apparent effect on DNA extension (Fig. 3a). Importantly, the point mutant Cdc13-2, which does not interact with Stn1 in vivo6, fails to repress telomerase activity with Stn1/Ten1 in vitro (Fig. 3b) indicating that the Stn1/Ten1 interaction is specific for wild type Cdc13. Our in vitro data support recent in vivo work suggesting Cdc13 and Stn1 function to block telomerase action at a telomere17.


The Hsp82 molecular chaperone promotes a switch between unextendable and extendable telomere states.

DeZwaan DC, Toogun OA, Echtenkamp FJ, Freeman BC - Nat. Struct. Mol. Biol. (2009)

Stn1 and Ten1 cooperate with Cdc13 to form an unextendable telomere protein-DNA complex in vitro. (a) The effect of an Stn1/Ten1 protein titration (50, 100, 250, 500 and 1000 nM) on telomerase DNA extension in the presence or absence of Cdc13 (250 nM) was determined using 23- or 7-base 3′-overhang DNAs. For comparison, the activity of unsupplemented and Cdc13 supplemented telomerase extract is shown. (b) Cdc13-2 does not form an unextendable DNA complex with Stn1/Ten1. Telomerase DNA extension reactions were performed using a 23-nucleotide 3′-overhang substrate and the reactions were supplemented with Cdc13 (FL) (250 nM) or Cdc13-2 (2) (250 nM) with or without Stn1/Ten1 (250 nM), as marked. All extension reactions were supplemented with a loading control primer (arrow) prior to precipitation and electrophoretic resolution and the +1 position for each DNA substrates is indicated. (c) The carboxyl-terminus of Cdc13 harbors an Stn1/Ten1 interaction surface. Fluorescence anisotropy with a fluorescein-labeled 15-base 3′-overhang telomeric oligonucleotide was used to detect DNA association; the 15-base DNA section provides a single Cdc13 binding site. The anisotropy was measured for each Cdc13 derivative (100 nM) alone and in the presence of Stn1/Ten1 (100 nM). The quantified data represent average values (mean +/− s.d.) from 5 independent assays.
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Figure 3: Stn1 and Ten1 cooperate with Cdc13 to form an unextendable telomere protein-DNA complex in vitro. (a) The effect of an Stn1/Ten1 protein titration (50, 100, 250, 500 and 1000 nM) on telomerase DNA extension in the presence or absence of Cdc13 (250 nM) was determined using 23- or 7-base 3′-overhang DNAs. For comparison, the activity of unsupplemented and Cdc13 supplemented telomerase extract is shown. (b) Cdc13-2 does not form an unextendable DNA complex with Stn1/Ten1. Telomerase DNA extension reactions were performed using a 23-nucleotide 3′-overhang substrate and the reactions were supplemented with Cdc13 (FL) (250 nM) or Cdc13-2 (2) (250 nM) with or without Stn1/Ten1 (250 nM), as marked. All extension reactions were supplemented with a loading control primer (arrow) prior to precipitation and electrophoretic resolution and the +1 position for each DNA substrates is indicated. (c) The carboxyl-terminus of Cdc13 harbors an Stn1/Ten1 interaction surface. Fluorescence anisotropy with a fluorescein-labeled 15-base 3′-overhang telomeric oligonucleotide was used to detect DNA association; the 15-base DNA section provides a single Cdc13 binding site. The anisotropy was measured for each Cdc13 derivative (100 nM) alone and in the presence of Stn1/Ten1 (100 nM). The quantified data represent average values (mean +/− s.d.) from 5 independent assays.
Mentions: In addition to Cdc13, Stn1 and Ten1 serve in telomere capping in vivo1. To determine whether FL-Cdc13 can function with Stn1 and Ten1 we purified the proteins (Supplementary Fig. 2a) and tested the effects of the proteins on telomerase activity. In the presence of Cdc13 we observed a striking reduction in DNA extension with increasing Stn1/Ten1 levels using the 23-base 3′-overhang DNA but not the 7-base substrate (Fig. 3a and Supplementary Fig. 2b). In the absence of Cdc13, Stn1/Ten1 had no apparent effect on DNA extension (Fig. 3a). Importantly, the point mutant Cdc13-2, which does not interact with Stn1 in vivo6, fails to repress telomerase activity with Stn1/Ten1 in vitro (Fig. 3b) indicating that the Stn1/Ten1 interaction is specific for wild type Cdc13. Our in vitro data support recent in vivo work suggesting Cdc13 and Stn1 function to block telomerase action at a telomere17.

Bottom Line: We have established an in vitro yeast telomere system in which Stn1-Ten1-unextendable or telomerase-extendable states can be observed.Both assemblies are Cdc13 dependent, as the Cdc13 C-terminal region supports Stn1-Ten1 interactions and the N-terminal region contains a telomerase-activation function.Notably, the yeast Hsp90 chaperone Hsp82 mediates the switch between the telomere capping and extending structures by modulating the DNA binding activity of Cdc13.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Developmental Biology, University of Illinois, Urbana-Champaign, Urbana, Illinois, USA.

ABSTRACT
Distinct protein assemblies are nucleated at telomeric DNA to both guard the ends from damage and lengthen the DNA after replication. In yeast, Cdc13 recruits either Stn1-Ten1 to form a protective cap or the telomerase holoenzyme to extend the DNA. We have established an in vitro yeast telomere system in which Stn1-Ten1-unextendable or telomerase-extendable states can be observed. Both assemblies are Cdc13 dependent, as the Cdc13 C-terminal region supports Stn1-Ten1 interactions and the N-terminal region contains a telomerase-activation function. Notably, the yeast Hsp90 chaperone Hsp82 mediates the switch between the telomere capping and extending structures by modulating the DNA binding activity of Cdc13. Taken together, our data show that the Hsp82 chaperone facilitates telomere DNA maintenance by promoting transitions between two operative complexes and by reducing the potential for binding events that would otherwise block the assembly of downstream structures.

Show MeSH
Related in: MedlinePlus