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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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Cdc13 stimulates telomerase DNA extension activity independent of single-stranded 3′-overhang DNA length. (a) The Cdc13 effect on telomerase-mediated DNA extension was determined using DNA substrates with either 23- or 7-nucleotide 3′-overhangs and a Cdc13 protein titration (50, 100, 250, 500 and 1000 nM), as marked. To control for possible non-specific protein affects the impact of BSA (B) (1000 nM) addition was tested. 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 marked. (b) Cdc13 can tether to DNA-bound telomerase. Fluorescence anisotropy with a fluorescein-labeled telomeric oligonucleotide was used to detect DNA association. The binding activities of Cdc13 (250 nM) or telomerase alone on 22- and 7-base DNA substrates were examined and the ability of Cdc13 to join DNA-bound telomerase was determined by adding purified Cdc13 (250 nM) to telomerase-DNA complexes using either the 22- and 7-base DNA substrates, as indicated. To demonstrate telomerase-dependent DNA binding RNaseA treatment was included19. The quantified data represent average values (mean +/− s.d.) from 5 independent assays.
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Figure 1: Cdc13 stimulates telomerase DNA extension activity independent of single-stranded 3′-overhang DNA length. (a) The Cdc13 effect on telomerase-mediated DNA extension was determined using DNA substrates with either 23- or 7-nucleotide 3′-overhangs and a Cdc13 protein titration (50, 100, 250, 500 and 1000 nM), as marked. To control for possible non-specific protein affects the impact of BSA (B) (1000 nM) addition was tested. 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 marked. (b) Cdc13 can tether to DNA-bound telomerase. Fluorescence anisotropy with a fluorescein-labeled telomeric oligonucleotide was used to detect DNA association. The binding activities of Cdc13 (250 nM) or telomerase alone on 22- and 7-base DNA substrates were examined and the ability of Cdc13 to join DNA-bound telomerase was determined by adding purified Cdc13 (250 nM) to telomerase-DNA complexes using either the 22- and 7-base DNA substrates, as indicated. To demonstrate telomerase-dependent DNA binding RNaseA treatment was included19. The quantified data represent average values (mean +/− s.d.) from 5 independent assays.

Mentions: As a first step we determined the Cdc13 effect on telomerase DNA extension activity in vitro. We anticipated that Cdc13 would inhibit telomerase function by competing for the DNA substrate, as observed for the human Cdc13 ortholog Pot113. To discriminate Cdc13 DNA binding-dependent effects we used DNA substrates with either 23- or 7-nucleotide 3′-overhangs in the telomerase extension assays. Prior biochemical studies indicated that Cdc13 requires a minimum of 11 single-stranded nucleotides to bind DNA14. In accordance, we found that our Cdc13 protein did not bind to DNA with a 7-base 3′-overhang but did bind to a 22-nucleotide overhang substrate (Supplementary Fig. 1). Unexpectedly, Cdc13 activated telomerase DNA extension activity independent of 3′-overhang DNA length (Fig. 1a). The DNA products below the +1 position for the 23-base substrate are likely produced by a previously described telomerase-associated endonuclease activity15.


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)

Cdc13 stimulates telomerase DNA extension activity independent of single-stranded 3′-overhang DNA length. (a) The Cdc13 effect on telomerase-mediated DNA extension was determined using DNA substrates with either 23- or 7-nucleotide 3′-overhangs and a Cdc13 protein titration (50, 100, 250, 500 and 1000 nM), as marked. To control for possible non-specific protein affects the impact of BSA (B) (1000 nM) addition was tested. 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 marked. (b) Cdc13 can tether to DNA-bound telomerase. Fluorescence anisotropy with a fluorescein-labeled telomeric oligonucleotide was used to detect DNA association. The binding activities of Cdc13 (250 nM) or telomerase alone on 22- and 7-base DNA substrates were examined and the ability of Cdc13 to join DNA-bound telomerase was determined by adding purified Cdc13 (250 nM) to telomerase-DNA complexes using either the 22- and 7-base DNA substrates, as indicated. To demonstrate telomerase-dependent DNA binding RNaseA treatment was included19. The quantified data represent average values (mean +/− s.d.) from 5 independent assays.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2744139&req=5

Figure 1: Cdc13 stimulates telomerase DNA extension activity independent of single-stranded 3′-overhang DNA length. (a) The Cdc13 effect on telomerase-mediated DNA extension was determined using DNA substrates with either 23- or 7-nucleotide 3′-overhangs and a Cdc13 protein titration (50, 100, 250, 500 and 1000 nM), as marked. To control for possible non-specific protein affects the impact of BSA (B) (1000 nM) addition was tested. 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 marked. (b) Cdc13 can tether to DNA-bound telomerase. Fluorescence anisotropy with a fluorescein-labeled telomeric oligonucleotide was used to detect DNA association. The binding activities of Cdc13 (250 nM) or telomerase alone on 22- and 7-base DNA substrates were examined and the ability of Cdc13 to join DNA-bound telomerase was determined by adding purified Cdc13 (250 nM) to telomerase-DNA complexes using either the 22- and 7-base DNA substrates, as indicated. To demonstrate telomerase-dependent DNA binding RNaseA treatment was included19. The quantified data represent average values (mean +/− s.d.) from 5 independent assays.
Mentions: As a first step we determined the Cdc13 effect on telomerase DNA extension activity in vitro. We anticipated that Cdc13 would inhibit telomerase function by competing for the DNA substrate, as observed for the human Cdc13 ortholog Pot113. To discriminate Cdc13 DNA binding-dependent effects we used DNA substrates with either 23- or 7-nucleotide 3′-overhangs in the telomerase extension assays. Prior biochemical studies indicated that Cdc13 requires a minimum of 11 single-stranded nucleotides to bind DNA14. In accordance, we found that our Cdc13 protein did not bind to DNA with a 7-base 3′-overhang but did bind to a 22-nucleotide overhang substrate (Supplementary Fig. 1). Unexpectedly, Cdc13 activated telomerase DNA extension activity independent of 3′-overhang DNA length (Fig. 1a). The DNA products below the +1 position for the 23-base substrate are likely produced by a previously described telomerase-associated endonuclease activity15.

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