We have isolated STNl, an essential Saccbaromyces cerevisiae gene, as a suppressor of the cdcl3-l mutation. A synthetic lethal interaction between a temperature-sensitive mutant allele of STNl, stnl-13, and cdcl3-l was observed. Stnl and Cdcl3 proteins displayed a physical interaction by two-hybrid analysis. As shown previously for cdcl3-l, stnl-13 cells at the restrictive temperature accumulate single-stranded DNA in subtelomeric regions of the chromosomes, but to a lesser extent than cdcl3-l cells. In addition, both Cdcl3 and Stnl were found to be involved in the regulation of telomere length, mutations in STNl or CDC13 conferring an increase in telomere size. Loss of Stnl function activated the RAD9 and MEC3 G2/M checkpoints, therefore confirming that DNA damage is generated. We propose that Stnl functions in telomere metabolism during late S phase in cooperation with Cdcl3.

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The Saccharomyces cerevisiae CDC13 protein binds single-strand telomeric DNA. Here we report the isolation of new mutant alleles of CDC13 that confer either abnormal telomere lengthening or telomere shortening. This deregulation not only depended on telomerase (Est2/TLC1) and Est1, a direct regulator of telomerase, but also on the yeast Ku proteins, yKu70/Hdf1 and yKu80/Hdf2, that have been previously implicated in DNA repair and telomere maintenance. Expression of a Cdc13-yKu70 fusion protein resulted in telomere elongation, similar to that produced by a Cdc13-Est1 fusion, thus suggesting that yKu70 might promote Cdc13-mediated telomerase recruitment. We also demonstrate that Stn1 is an inhibitor of telomerase recruitment by Cdc13, based both on STN1 overexpression and Cdc13-Stn1 fusion experiments. We propose that accurate regulation of telomerase recruitment by Cdc13 results from a coordinated balance between positive control by yKu70 and negative control by Stn1. Our results represent the first evidence of a direct control of the telomerase-loading function of Cdc13 by a double-strand telomeric DNA-binding complex.Telomeres, the ends of eukaryotic chromosomes, are critical for maintaining chromosome stability and genome integrity (2,8,60). Telomeres are composed of particular DNA sequences which are rich in TG and arranged in species-specific repeated motifs. Telomeres are capped by proteins that bind to these repeating DNA sequences (6,20). This apparently serves at least two distinct purposes. First, some of these telomeric proteins presumably form complexes that regulate telomerase activity and, hence, the length of telomeric tracts (31,43). Some telomeric proteins have also been implicated in the physical protection of chromosome ends (38), in preventing recombinational events that would otherwise frequently occur between repeating telomeric sequences (33,34,53), and in keeping off DNA repair enzymes (14). Indeed, telomeres represent naturally occurring DNA double-strand breaks that, contrary to those resulting from accidental damage, do not need to (and must not) be repaired. Surprisingly, however, yeast Ku proteins, as well as proteins of the Mre11-Rad50-Xrs2 complex, which have been implicated in DNA repair by nonhomologous end joining have also been implicated in telomere maintenance (3,4,7,11,12,27,28,39,44,46,47). Moreover, yKu70 and yKu80 have been found to localize at the telomeres (18,37).The repeating TG-rich telomeric DNA sequences are mostly double stranded. However, during S phase only, telomeres display a short (ca. 35-to 50-nucleotide) single-stranded DNA extension that marks the very end of the telomere (57, 58). Single-stranded telomeric DNA is thought to represent the site of anchoring of telomerase, which is composed of the evolutionary conserved Est2 reverse transcriptase enzyme and of the TLC1 RNA template (31, 42). However, recent experiments suggest that telomerase-dependent elongation of de novo ends does not appear to involve single strandedness and does not require significant d...

BackgroundThe exact role of primary nanoparticle (NP) size and their degree of agglomeration in aerosols on the determination of pulmonary effects is still poorly understood. Smaller NP are thought to have greater biological reactivity, but their level of agglomeration in an aerosol may also have an impact on pulmonary response. The aim of this study was to investigate the role of primary NP size and the agglomeration state in aerosols, using well-characterized TiO2 NP, on their relative pulmonary toxicity, through inflammatory, cytotoxic and oxidative stress effects in Fisher 344 male rats.MethodsThree different sizes of TiO2 NP, i.e., 5, 10–30 or 50 nm, were inhaled as small (SA) (< 100 nm) or large agglomerates (LA) (> 100 nm) at 20 mg/m3 for 6 hours.ResultsCompared to the controls, bronchoalveolar lavage fluids (BALF) showed that LA aerosols induced an acute inflammatory response, characterized by a significant increase in the number of neutrophils, while SA aerosols produced significant oxidative stress damages and cytotoxicity. Data also demonstrate that for an agglomeration state smaller than 100 nm, the 5 nm particles caused a significant increase in cytotoxic effects compared to controls (assessed by an increase in LDH activity), while oxidative damage measured by 8-isoprostane concentration was less when compared to 10–30 and 50 nm particles. In both SA and LA aerosols, the 10–30 nm TiO2 NP size induced the most pronounced pro-inflammatory effects compared to controls.ConclusionsOverall, this study showed that initial NP size and agglomeration state are key determinants of nano-TiO2 lung inflammatory reaction, cytotoxic and oxidative stress induced effects.