The finger subdomain of yeast telomerase cooperates with Pif1p to limit telomere elongation.

Telomere synthesis depends on telomerase, which contains an RNA subunit linked to a specialized reverse transcriptase subunit and several associated proteins. Here we report the characterization of four mutations in the yeast reverse transcriptase subunit Est2p that cause an overelongation of telomeres and an increase in the association of Est1p with telomeres during S phase. These 'up-mutations' are clustered in the finger subdomain of the reverse transcriptase. We show that the catalytic properties of the up-mutant telomerases are not improved in vitro. In vivo, the up-mutations neither bypass the activation step governed by Cdc13p nor do they uncouple telomerase from the Rap1p inhibition pathway. In the presence of the up-mutations, however, the ability of the Pif1p helicase to decrease telomere length and to inhibit the association of Est1p with telomeres is impaired. In addition, Pif1p associates in vivo with the telomerase RNA (TLC1) in a way that depends on the finger subdomain. We propose that, in addition to its catalytic role, the finger subdomain of Est2p facilitates the action of Pif1p at telomeres.

Accurate repair of non-cohesive, double strand breaks in Saccharomyces cerevisiae: enhancement by homology-assisted end-joining.

Although the joining of blunt ends in yeast by non-homologous end joining (NHEJ) is reported to be inefficient in comparison to cohesive-end joining (Boulton and Jackson, 1996), we find that efficiency varies greatly, depending on strain, growth phase and sequence. In particular, the levels of efficiency of recircularization of a plasmid linearized by non-cohesive cleavage is augmented to that of cohesive end joining if the cleavage cut site is flanked by sequences present in the genome. We call this enhancement 'homology-assisted end joining' (HAEJ), which depends on components of the NHEJ repair pathway and, in some cases, on components of the homologous recombination (HR) pathway and on Htl1 a component of the remodels structure of chromatin (RSC) complex. The homologous genome sequences are not used as templates for repair DNA synthesis, but may facilitate end-to-end collision and ligation by providing a track for guided diffusion.

A study of biochemical and functional interactions of Htl1p, a putative component of the Saccharomyces cerevisiae, Rsc chromatin-remodeling complex.

HTL1, a small gene of Saccharomyces cerevisiae, encodes a 78-aminoacid peptide that influences the performance of a wide range of cellular processes [Lanzuolo, C., Ederle, S., Pollice, A., Russo, F., Storlazzi, A., Pulitzer, J.F., 2001. The HTL1 gene,YCR020W-b of Saccharomyces cerevisiae is necessary for growth at 37 degrees C, and for the conservation of chromosome stability and fertility. Yeast, 18, 1317-1330]. Genetic interactions and co-immunoprecipitation experiments indicate a role for Htl1p in functions controlled by RSC, a multiprotein, ATP-dependent, chromatin-remodeling complex [Lu, Y.M., Lin, Y.R., Tsai, A., Hsao, Y.S., Li, C.C., Cheng, M.Y., 2003. Dissecting the pet18 mutation in Saccharomyces cerevisiae: HTL1 encodes a 7-kDa polypeptide that interacts with components of the RSC complex. Mol. Genet. Genomics., 269, 321-330] [Romeo, M.J., Angus-Hill, M.L., Sobering, A.K., Kamada, Y., Cairns, B.R., Levin, D.E., 2002. HTL1 encodes a novel factor that interacts with the Rsc chromatin-remodeling complex in Saccharomyces cerevisiae. Mol. Cell. Biol., 22, 8165-8174]. Htl1p and RSC components, share the property of associating with TBP a component of general multiprotein transcription factor TFIID [Sanders, S.L., Jennings, J., Canutescu, A., Link, A.J., Weil, P.A., 2002. Proteomics of the eukaryotic transcription machinery: identification of proteins associated with components of yeast TFIID by multidimensional mass spectrometry. Mol. Cell. Biol. 22, 4723-4738]. We confirm, by integrating genetic and biochemical experiments, that Htl1p binding to the RSC complex is direct and physiologically relevant and show that it is mediated by Rsc8p, a core component of the RSC complex. Deletion of HTL1, like depletion of RSC core subunits [Moreira, J.M., Holmberg, S., 1999. Transcriptional repression of the yeast CHA1 gene requires the chromatin-remodeling complex Rsc. Embo J., 18, 2836-2844], leads to constitutive transcription of the CHA1 locus. This transcriptional phenotype exhibits variable penetrance. Deletion of HTL1 also leads to hydroxyurea hypersensitivity at 30 degrees C, suggesting a defect in replication/repair. This defect leads, during cell growth, to selection of mutations at the SIR3 locus that suppress hydroxyurea sensitivity.

Human telomeric position effect is determined by chromosomal context and telomeric chromatin integrity.

We investigated the influence of telomere proximity and composition on the expression of an EGFP reporter gene in human cells. In transient transfection assays, telomeric DNA does not repress EGFP but rather slightly increases its expression. In contrast, in stable cell lines, the same reporter construct is repressed when inserted at a subtelomeric location. The telomeric repression is transiently alleviated by increasing the dosage of the TTAGGG repeat factor 1 (TRF1). Upon a prolongated treatment with trichostatin A, the derepression of the subtelomeric reporter gene correlates with the delocalization of HP1alpha and HP1beta. In contrast, treating the cells with 5 azacytidin, a demethylating agent, or with sirtinol, an inhibitor of the Sir2 family of deacetylase, has no apparent effect on telomeric repression. Overall, position effects at human chromosome ends are dependent on a specific higher-order organization of the telomeric chromatin. The possible involvement of HP1 isoforms is discussed.