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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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Hsp82 promotes conversion of the Cdc13-capping structure into a Cdc13-extending complex by dissociating Cdc13 from DNA. (a) The ability of Hsp82 to convert an unextendable Cdc13/Stn1/Ten1-capped DNA complex into a telomerase accessible DNA structure was determined using a 23-base 3′-overhang DNA substrate, telomerase extract and an Hsp82 protein titration (0.25, 0.5, 1.0, 2.5, 5.0, 10.0 and 20.0 μM). The cellular Hsp82 concentration is 17 μM34. (b) Hsp82 dissociates amino-terminal Cdc13 protein derivates from DNA. Cdc13 DNA binding was monitored by fluorescence anisotropy. The impact of Hsp82 on the binding activities of FL, C, N or DBD Cdc13 protein derivatives (100 nM) was determined along with the effect of Sba1 on FL, as indicated.
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Figure 4: Hsp82 promotes conversion of the Cdc13-capping structure into a Cdc13-extending complex by dissociating Cdc13 from DNA. (a) The ability of Hsp82 to convert an unextendable Cdc13/Stn1/Ten1-capped DNA complex into a telomerase accessible DNA structure was determined using a 23-base 3′-overhang DNA substrate, telomerase extract and an Hsp82 protein titration (0.25, 0.5, 1.0, 2.5, 5.0, 10.0 and 20.0 μM). The cellular Hsp82 concentration is 17 μM34. (b) Hsp82 dissociates amino-terminal Cdc13 protein derivates from DNA. Cdc13 DNA binding was monitored by fluorescence anisotropy. The impact of Hsp82 on the binding activities of FL, C, N or DBD Cdc13 protein derivatives (100 nM) was determined along with the effect of Sba1 on FL, as indicated.

Mentions: The ability of the DBD and C-domain to interfere with DNA extension indicates that Cdc13 DNA binding activity is sufficient to inhibit telomerase, yet full-length Cdc13 only activates, unless Stn1/Ten1 is also supplemented. To account for these results we believed that a telomerase cofactor was present in the reactions, preventing Cdc13 from interfering with telomerase but still permitting DNA-bound telomerase to associate with Cdc13. Given the established Hsp82 roles with capping and extending telomere components, we tested whether Hsp82 might affect Cdc13. As an initial test Hsp82 was titrated into DNA extension reactions containing a Cdc13/Stn1/Ten1-capped telomeric DNA substrate (Fig. 4a). In the absence of Supplementary Hsp82, Cdc13/Stn1/Ten1 suppressed the DNA extension activity below basal levels (Fig. 4a lanes 1 & 3). However, in an Hsp82-dependent manner the extension activity increased ~15-fold, which indicates that Hsp82 is sufficient to switch the Cdc13/Stn1/Ten1-capping structure into a Cdc13/telomerase-extending complex (Fig. 4a).


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)

Hsp82 promotes conversion of the Cdc13-capping structure into a Cdc13-extending complex by dissociating Cdc13 from DNA. (a) The ability of Hsp82 to convert an unextendable Cdc13/Stn1/Ten1-capped DNA complex into a telomerase accessible DNA structure was determined using a 23-base 3′-overhang DNA substrate, telomerase extract and an Hsp82 protein titration (0.25, 0.5, 1.0, 2.5, 5.0, 10.0 and 20.0 μM). The cellular Hsp82 concentration is 17 μM34. (b) Hsp82 dissociates amino-terminal Cdc13 protein derivates from DNA. Cdc13 DNA binding was monitored by fluorescence anisotropy. The impact of Hsp82 on the binding activities of FL, C, N or DBD Cdc13 protein derivatives (100 nM) was determined along with the effect of Sba1 on FL, as indicated.
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Related In: Results  -  Collection

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Figure 4: Hsp82 promotes conversion of the Cdc13-capping structure into a Cdc13-extending complex by dissociating Cdc13 from DNA. (a) The ability of Hsp82 to convert an unextendable Cdc13/Stn1/Ten1-capped DNA complex into a telomerase accessible DNA structure was determined using a 23-base 3′-overhang DNA substrate, telomerase extract and an Hsp82 protein titration (0.25, 0.5, 1.0, 2.5, 5.0, 10.0 and 20.0 μM). The cellular Hsp82 concentration is 17 μM34. (b) Hsp82 dissociates amino-terminal Cdc13 protein derivates from DNA. Cdc13 DNA binding was monitored by fluorescence anisotropy. The impact of Hsp82 on the binding activities of FL, C, N or DBD Cdc13 protein derivatives (100 nM) was determined along with the effect of Sba1 on FL, as indicated.
Mentions: The ability of the DBD and C-domain to interfere with DNA extension indicates that Cdc13 DNA binding activity is sufficient to inhibit telomerase, yet full-length Cdc13 only activates, unless Stn1/Ten1 is also supplemented. To account for these results we believed that a telomerase cofactor was present in the reactions, preventing Cdc13 from interfering with telomerase but still permitting DNA-bound telomerase to associate with Cdc13. Given the established Hsp82 roles with capping and extending telomere components, we tested whether Hsp82 might affect Cdc13. As an initial test Hsp82 was titrated into DNA extension reactions containing a Cdc13/Stn1/Ten1-capped telomeric DNA substrate (Fig. 4a). In the absence of Supplementary Hsp82, Cdc13/Stn1/Ten1 suppressed the DNA extension activity below basal levels (Fig. 4a lanes 1 & 3). However, in an Hsp82-dependent manner the extension activity increased ~15-fold, which indicates that Hsp82 is sufficient to switch the Cdc13/Stn1/Ten1-capping structure into a Cdc13/telomerase-extending complex (Fig. 4a).

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