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Telomerase RNA Template Mutations Reveal Sequence-Specific Requirements for the Activation and Repression of Telomerase Action at Telomeres

Telomerase RNA Template Mutations Reveal Sequence-Specific Requirements for the Activation and... Telomerase RNA Template Mutations Reveal Sequence-Specific Requirements for the Activation and Repression of Telomerase Action at Telomeres John C. Prescott † and Elizabeth H. Blackburn * Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, California 94143-0414 ABSTRACT Telomeric DNA is maintained within a length range characteristic of an organism or cell type. Significant deviations outside this range are associated with altered telomere function. The yeast telomere-binding protein Rap1p negatively regulates telomere length. Telomere elongation is responsive to both the number of Rap1p molecules bound to a telomere and the Rap1p-centered DNA-protein complex at the extreme telomeric end. Previously, we showed that a specific trinucleotide substitution in the Saccharomyces cerevisiae telomerase gene ( TLC1 ) RNA template abolished the enzymatic activity of telomerase, causing the same cell senescence and telomere shortening phenotypes as a complete tlc1 deletion. Here we analyze effects of six single- and double-base changes within these same three positions. All six mutant telomerases had in vitro enzymatic activity levels similar to the wild-type levels. The base changes predicted from the mutations all disrupted Rap1p binding in vitro to the corresponding duplex DNAs. However, they caused two classes of effects on telomere homeostasis: (i) rapid, RAD52-independent telomere lengthening and poor length regulation, whose severity correlated with the decrease in in vitro Rap1p binding affinity (this is consistent with loss of negative regulation of telomerase action at these telomeres; and (ii) telomere shortening that, depending on the template mutation, either established a new short telomere set length with normal cell growth or was progressive and led to cellular senescence. Hence, disrupting Rap1p binding at the telomeric terminus is not sufficient to deregulate telomere elongation. This provides further evidence that both positive and negative cis -acting regulators of telomerase act at telomeres. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Molecular and Cellular Biology American Society For Microbiology

Telomerase RNA Template Mutations Reveal Sequence-Specific Requirements for the Activation and Repression of Telomerase Action at Telomeres

Telomerase RNA Template Mutations Reveal Sequence-Specific Requirements for the Activation and Repression of Telomerase Action at Telomeres

Molecular and Cellular Biology , Volume 20 (8): 2941 – Apr 15, 2000

Abstract

Telomerase RNA Template Mutations Reveal Sequence-Specific Requirements for the Activation and Repression of Telomerase Action at Telomeres John C. Prescott † and Elizabeth H. Blackburn * Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, California 94143-0414 ABSTRACT Telomeric DNA is maintained within a length range characteristic of an organism or cell type. Significant deviations outside this range are associated with altered telomere function. The yeast telomere-binding protein Rap1p negatively regulates telomere length. Telomere elongation is responsive to both the number of Rap1p molecules bound to a telomere and the Rap1p-centered DNA-protein complex at the extreme telomeric end. Previously, we showed that a specific trinucleotide substitution in the Saccharomyces cerevisiae telomerase gene ( TLC1 ) RNA template abolished the enzymatic activity of telomerase, causing the same cell senescence and telomere shortening phenotypes as a complete tlc1 deletion. Here we analyze effects of six single- and double-base changes within these same three positions. All six mutant telomerases had in vitro enzymatic activity levels similar to the wild-type levels. The base changes predicted from the mutations all disrupted Rap1p binding in vitro to the corresponding duplex DNAs. However, they caused two classes of effects on telomere homeostasis: (i) rapid, RAD52-independent telomere lengthening and poor length regulation, whose severity correlated with the decrease in in vitro Rap1p binding affinity (this is consistent with loss of negative regulation of telomerase action at these telomeres; and (ii) telomere shortening that, depending on the template mutation, either established a new short telomere set length with normal cell growth or was progressive and led to cellular senescence. Hence, disrupting Rap1p binding at the telomeric terminus is not sufficient to deregulate telomere elongation. This provides further evidence that both positive and negative cis -acting regulators of telomerase act at telomeres.

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Publisher
American Society For Microbiology
Copyright
Copyright © 2000 by the American society for Microbiology.
ISSN
0270-7306
eISSN
1098-5549
DOI
10.1128/MCB.20.8.2941-2948.2000
Publisher site
See Article on Publisher Site

Abstract

Telomerase RNA Template Mutations Reveal Sequence-Specific Requirements for the Activation and Repression of Telomerase Action at Telomeres John C. Prescott † and Elizabeth H. Blackburn * Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, California 94143-0414 ABSTRACT Telomeric DNA is maintained within a length range characteristic of an organism or cell type. Significant deviations outside this range are associated with altered telomere function. The yeast telomere-binding protein Rap1p negatively regulates telomere length. Telomere elongation is responsive to both the number of Rap1p molecules bound to a telomere and the Rap1p-centered DNA-protein complex at the extreme telomeric end. Previously, we showed that a specific trinucleotide substitution in the Saccharomyces cerevisiae telomerase gene ( TLC1 ) RNA template abolished the enzymatic activity of telomerase, causing the same cell senescence and telomere shortening phenotypes as a complete tlc1 deletion. Here we analyze effects of six single- and double-base changes within these same three positions. All six mutant telomerases had in vitro enzymatic activity levels similar to the wild-type levels. The base changes predicted from the mutations all disrupted Rap1p binding in vitro to the corresponding duplex DNAs. However, they caused two classes of effects on telomere homeostasis: (i) rapid, RAD52-independent telomere lengthening and poor length regulation, whose severity correlated with the decrease in in vitro Rap1p binding affinity (this is consistent with loss of negative regulation of telomerase action at these telomeres; and (ii) telomere shortening that, depending on the template mutation, either established a new short telomere set length with normal cell growth or was progressive and led to cellular senescence. Hence, disrupting Rap1p binding at the telomeric terminus is not sufficient to deregulate telomere elongation. This provides further evidence that both positive and negative cis -acting regulators of telomerase act at telomeres.

Journal

Molecular and Cellular BiologyAmerican Society For Microbiology

Published: Apr 15, 2000

References