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Molecular and Cellular Biology, April 2000, p. 2941-2948, Vol. 20, No. 8
Department of Microbiology and Immunology,
University of California, San Francisco, San Francisco, California
94143-0414
Received 19 October 1999/Returned for modification 17 November
1999/Accepted 19 January 2000
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.
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Telomerase RNA Template Mutations Reveal Sequence-Specific
Requirements for the Activation and Repression of Telomerase Action
at Telomeres
and
*
Corresponding author. Mailing address: Phone: (415)
476-4912. Fax: (415) 476-8201. E-mail:
telomer{at}itsa.ucsf.edu.
Present address: Sunesis Pharmaceuticals, Redwood City, Calif.
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