175
views
0
recommends
+1 Recommend
0 collections
    4
    shares
      • Record: found
      • Abstract: found
      • Article: found
      Is Open Access

      The Saccharomyces cerevisiae Telomerase Subunit Est3 Binds Telomeres in a Cell Cycle– and Est1–Dependent Manner and Interacts Directly with Est1 In Vitro

      research-article
      , , , *
      PLoS Genetics
      Public Library of Science

      Read this article at

      Bookmark
          There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.

          Abstract

          Telomerase is a telomere dedicated reverse transcriptase that replicates the very ends of eukaryotic chromosomes. Saccharomyces cerevisiae telomerase consists of TLC1 (the RNA template), Est2 (the catalytic subunit), and two accessory proteins, Est1 and Est3, that are essential in vivo for telomerase activity but are dispensable for catalysis in vitro. Est1 functions in both recruitment and activation of telomerase. The association of Est3 with telomeres occurred largely in late S/G2 phase, the time when telomerase acts and Est1 telomere binding occurs. Est3 telomere binding was Est1-dependent. This dependence is likely due to a direct interaction between the two proteins, as purified recombinant Est1 and Est3 interacted in vitro. Est3 abundance was neither cell cycle–regulated nor Est1-dependent. Est3 was the most abundant of the three Est proteins (84.3±13.3 molecules per cell versus 71.1±19.2 for Est1 and 37.2±6.5 for Est2), so its telomere association and/or activity is unlikely to be limited by its relative abundance. Est2 and Est1 telomere binding was unaffected by the absence of Est3. Taken together, these data indicate that Est3 acts downstream of both Est2 and Est1 and that the putative activation function of Est1 can be explained by its role in recruiting Est3 to telomeres.

          Author Summary

          Owing to the biochemical properties of DNA polymerases, the free ends of linear chromosomes, called telomeres, cannot be replicated by the same mechanisms that suffice for the rest of the chromosome. Instead they are maintained by a telomere-dedicated reverse transcriptase called telomerase that uses its integral RNA component as the template to make more telomeric DNA. In baker's yeast, telomerase is composed of a catalytic subunit (Est2), the templating RNA (TLC1), and two accessory proteins, Est1 and Est3. Here we show that Est3 associates with telomeres late in the cell cycle, at the same time when telomerase is active, and this binding was Est1-dependent, even though Est3 abundance was neither cell cycle–regulated nor Est1-dependent. Since purified Est3 and Est1interacted in vitro, Est1-dependent recruitment of Est3 is probably due to direct protein–protein interaction. Neither Est1 nor Est2 telomere binding was Est3-dependent. Thus, Est3 acts downstream of telomerase recruitment to promote telomerase activity, and the telomerase activation functions of Est1 can be explained by its recruiting Est3 to telomeres.

          Related collections

          Most cited references45

          • Record: found
          • Abstract: found
          • Article: not found

          Single-stranded DNA arising at telomeres in cdc13 mutants may constitute a specific signal for the RAD9 checkpoint.

          A cdc13 temperature-sensitive mutant of Saccharomyces cerevisiae arrests in the G2 phase of the cell cycle at the restrictive temperature as a result of DNA damage that activates the RAD9 checkpoint. The DNA lesions present after a failure of Cdc13p function appear to be located almost exclusively in telomere-proximal regions, on the basis of the profile of induced mitotic recombination. cdc13 rad9 cells dividing at the restrictive temperature contain single-stranded DNA corresponding to telomeric and telomere-proximal DNA sequences and eventually lose telomere-associated sequences. These results suggest that the CDC13 product functions in telomere metabolism, either in the replication of telomeric DNA or in protecting telomeres from the double-strand break repair system. Moreover, since cdc13 rad9 cells divide at a wild-type rate for several divisions at the restrictive temperature while cdc13 RAD9 cells arrest in G2, these results also suggest that single-stranded DNA may be a specific signal for the RAD9 checkpoint.
            Bookmark
            • Record: found
            • Abstract: found
            • Article: not found

            TLC1: template RNA component of Saccharomyces cerevisiae telomerase.

            Telomeres, the natural ends of linear eukaryotic chromosomes, are essential for chromosome stability. Because of the nature of DNA replication, telomeres require a specialized mechanism to ensure their complete duplication. Telomeres are also capable of silencing the transcription of genes that are located near them. In order to identify genes in the budding yeast Saccharomyces cerevisiae that are important for telomere function, a screen was conducted for genes that, when expressed in high amounts, would suppress telomeric silencing. This screen lead to the identification of the gene TLC1 (telomerase component 1). TLC1 encodes the template RNA of telomerase, a ribonucleoprotein required for telomere replication in a variety of organisms. The discovery of TLC1 confirms the existence of telomerase in S. cerevisiae and may facilitate both the analysis of this enzyme and an understanding of telomere structure and function.
              Bookmark
              • Record: found
              • Abstract: found
              • Article: not found

              Homologous chromosome interactions in meiosis: diversity amidst conservation.

              Proper chromosome segregation is crucial for preventing fertility problems, birth defects and cancer. During mitotic cell divisions, sister chromatids separate from each other to opposite poles, resulting in two daughter cells that each have a complete copy of the genome. Meiosis poses a special problem in which homologous chromosomes must first pair and then separate at the first meiotic division before sister chromatids separate at the second meiotic division. So, chromosome interactions between homologues are a unique feature of meiosis and are essential for proper chromosome segregation. Pairing and locking together of homologous chromosomes involves recombination interactions in some cases, but not in others. Although all organisms must match and lock homologous chromosomes to maintain genome integrity throughout meiosis, recent results indicate that the underlying mechanisms vary in different organisms.
                Bookmark

                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Genet
                plos
                plosgen
                PLoS Genetics
                Public Library of Science (San Francisco, USA )
                1553-7390
                1553-7404
                May 2011
                May 2011
                5 May 2011
                : 7
                : 5
                : e1002060
                Affiliations
                [1]Department of Molecular Biology, Princeton University, Princeton, New Jersey, United States of America
                Fred Hutchinson Cancer Research Center, United States of America
                Author notes

                Conceived and designed the experiments: CTT YW VAZ. Performed the experiments: CTT YW AC. Analyzed the data: CTT YW AC. Contributed reagents/materials/analysis tools: CTT YW AC. Wrote the paper: CTT YW AC VAZ.

                Article
                PGENETICS-D-11-00235
                10.1371/journal.pgen.1002060
                3088721
                21573139
                3ad16463-535e-4bad-9929-855a5bebec26
                Tuzon et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
                History
                : 31 January 2011
                : 2 March 2011
                Page count
                Pages: 12
                Categories
                Research Article
                Biology
                Biochemistry
                Biomacromolecule-Ligand Interactions
                Genetics
                Gene Function
                Molecular Genetics
                Molecular Cell Biology
                Cell Division
                Nucleic Acids

                Genetics
                Genetics

                Comments

                Comment on this article