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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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The amino-terminal domain of Cdc13 has a telomerase-activation function. The effect of full-length (FL) Cdc13, Cdc13-2 (2), amino-terminus (N), carboxyl-terminus (C) or DNA binding domain (DBD) proteins (250 nM) on telomerase DNA extension using 7- or 23-base 3′-overhang DNA substrates was determined. 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.
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Figure 2: The amino-terminal domain of Cdc13 has a telomerase-activation function. The effect of full-length (FL) Cdc13, Cdc13-2 (2), amino-terminus (N), carboxyl-terminus (C) or DNA binding domain (DBD) proteins (250 nM) on telomerase DNA extension using 7- or 23-base 3′-overhang DNA substrates was determined. 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.

Mentions: To better understand how Cdc13 regulates telomerase activity we produced and characterized a series of Cdc13 protein derivatives that included full-length (FL), Cdc13-2 point mutant (2), DNA binding domain (DBD), amino-terminus through the DBD (N) and DBD through the carboxyl-terminus (C) (Fig. 2); all derivatives displayed high-affinity DNA binding activity (data not shown). The FL, Cdc13-2 and the N-fragment activated telomerase DNA extension activity on either DNA substrate. Hence, in addition to its suggested telomere recruitment function6, the Cdc13 N-terminus has a telomerase activation surface. In contrast, the C or DBD derivatives inhibited extension of the 23-base substrate but did not affect basal extension of the 7-base 3′-overhang DNA (Fig. 2).


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)

The amino-terminal domain of Cdc13 has a telomerase-activation function. The effect of full-length (FL) Cdc13, Cdc13-2 (2), amino-terminus (N), carboxyl-terminus (C) or DNA binding domain (DBD) proteins (250 nM) on telomerase DNA extension using 7- or 23-base 3′-overhang DNA substrates was determined. 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.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: The amino-terminal domain of Cdc13 has a telomerase-activation function. The effect of full-length (FL) Cdc13, Cdc13-2 (2), amino-terminus (N), carboxyl-terminus (C) or DNA binding domain (DBD) proteins (250 nM) on telomerase DNA extension using 7- or 23-base 3′-overhang DNA substrates was determined. 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.
Mentions: To better understand how Cdc13 regulates telomerase activity we produced and characterized a series of Cdc13 protein derivatives that included full-length (FL), Cdc13-2 point mutant (2), DNA binding domain (DBD), amino-terminus through the DBD (N) and DBD through the carboxyl-terminus (C) (Fig. 2); all derivatives displayed high-affinity DNA binding activity (data not shown). The FL, Cdc13-2 and the N-fragment activated telomerase DNA extension activity on either DNA substrate. Hence, in addition to its suggested telomere recruitment function6, the Cdc13 N-terminus has a telomerase activation surface. In contrast, the C or DBD derivatives inhibited extension of the 23-base substrate but did not affect basal extension of the 7-base 3′-overhang DNA (Fig. 2).

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