Est1 and Cdc13 as comediators of telomerase access

Science

Volumn 286, Issue 5437, 1999, Pages 117-120

  Evans, Sara K ^a   Lundblad, Victoria ^a  

^a BAYLOR COLLEGE OF MEDICINE   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

HYBRID PROTEIN; TELOMERASE;

ARTICLE; CHROMATIN STRUCTURE; CONTROLLED STUDY; DNA BINDING; ENZYME ACTIVITY; ENZYME ANALYSIS; ENZYME BINDING; ENZYME SUBUNIT; NONHUMAN; PRIORITY JOURNAL; PROTEIN DOMAIN; SACCHAROMYCES CEREVISIAE; TELOMERE;

BINDING SITES; CYCLIN B; DNA, FUNGAL; DNA, SINGLE-STRANDED; FUNGAL PROTEINS; GENETIC COMPLEMENTATION TEST; HOMEOSTASIS; MODELS, BIOLOGICAL; MUTATION; RECOMBINANT FUSION PROTEINS; SACCHAROMYCES CEREVISIAE; SACCHAROMYCES CEREVISIAE PROTEINS; TELOMERASE; TELOMERE;

FUNGI; MYXOGASTRIA; SACCHAROMYCES CEREVISIAE;

EID: 0033214013     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.286.5437.117     Document Type: Article

References (30)

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17. 2-21,452-693. Telomere elongation by any of these fusion proteins is not due to increased protein expression in the context of the fusioǹ, because overexpression of Est1, Est2, or Cdc13, or expression of Est1 by the CDC13 promoter, has little or no effect on telomere length (8, 11, 17, 19).
18. We have also observed up to a ∼4-kb increase in the length of a single telomere (chromosome IIIL). Strains with greatly elongated telomeres do not exhibit any discernable growth defect but were not examined for more subtle defects (such as alterations in cell cycle progression). We have not investigated yet whether the increase in telomere length in strains carrying Cdc13-telomerase fusions occurs at a constant rate, or if there is some influence of cis-inhibition on elongation, as previously observed (3).
19. S. K. Evans and V. Lundblad, unpublished data.
20. The est1-47 mutation is one of a panel of alanine-scan mutations in EST1 (17); the Est1-47 mutant protein still retains association with telomerase (at 20% of WT levels), as assessed by coimmunoprecipitation with the TLC1 RNA (27).
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22. A. Chandra, T. R. Hughes, V. Lundblad, unpublished data.
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24. T. R. Hughes, R. G. Weilbaecher, V. Lundblad, in preparation.
25. D67OA fusion failed to complement an est2-Δ strain.
26. Bypass of est1-Δ senescence was not simply a consequence of increased Est2 levels (due to possible minimal increase in expression of EST2 by the CDC13 promoter), because even higher level expression of EST2 by the constitutive ADH promoter (8) was not sufficient to allow an est1-Δ strain to grow (17).
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28. Association of Cdc13-Est2 fusion protein with the TLC1 RNA was reduced by less than twofold in the absence of Est1, as assessed by immunoprecipitation (27), arguing that the failure to elongate telomeres in an est1-Δ strain is not simply due to reduced stability of the Cdc13-Est2 telomerase complex.
29. 2, 5% glycerol, 1 mM phenylmethylsulfonyl fluoride. 300 mM NaCl]. Cell extracts were prepared by five repeated cycles of freezing and grinding in liquid nitrogen. Extracts were cleared twice by centrifugation for 10 min at 14,000 rpm at 4°C and immunoprecipitated with an antibody to hemagglutinin (HA) (16β12, Babco) and protein A/G agarose beads (Calbiochem). RNA was prepared by SDS-phenol-chloroform extraction, and TLC1 was detected on 7 M urea-4% polyacrylamide gel as described (8). The efficiency of TLC1 recovery in immunoprecipitates is typically less than 2%; the recovery with untagged proteins was less than 0.05%.
30. We thank T. Hughes, R. Weilbaecher, and L. Zumstein for critical review of the manuscript. Supported by NIH grant RO1GM55867 (V.L.) and by a U.S. Army Breast Cancer Research Predoctoral Traineeship (S.K.E.).