A study on telomere protection and telomerase- and cap-independent mechanisms of telomere maintenance in yeast Saccharomyces cerevisiae .

摘要

An SGA approach to discover cdc13-1ts supressors . Telomeres, the DNA-protein complexes at the end of eukaryotic chromosomes, are essential for chromosomal stability. In yeast, the telomeric single-strand binding protein Cdc13p has multiple important roles related to telomere maintenance: (1) telomeric "capping"---protection of telomeres by forming complexes with yKu70/80 and with Stn1p/Ten1p; (2) positive regulation of telomere replication via interaction with Est1p, which is a part of telomerase; (3) negative regulation of telomerase by the recruitment of telomere elongation suppressors Stn1p and Ten1p.;Based on the comparative analysis of growth properties of the strains (23°C vs 33°C), the initial screen identified up to 111 genes that displayed an apparent growth at 33°C. In order to verify these results, diploids were regenerated, sporulated, microdissected, and haploid double mutants cdc13-1 yfgDelta were isolated from 38 potential cdc13-1 suppressors. Unfortunately, this verification failed to reproduce a suppression of the growth defect by any of the selected genes at any temperature. While disappointing, the results reemphasize that careful re-examination of large scale SGA approaches are indispensable before going on to more involved experimentation.;Similarities and differences between adaptation to DNA double-strand break and to telomere uncapping in yeast Saccharomyces cerevisiae. It was previously shown that a certain proportion of telomerase negative survivor cells (both type I and type II cells) is able to survive in the absence of the telomere capping protein Cdc13p. These strains (named Delta13s) were characterized in great detail and one of their discovered features was a striking ability to continuously inactivate DNA-damage checkpoints. Based on structural similarities between DNA double strand breaks (DSB) and unprotected telomeres, we attempted to verify if the molecular mechanisms regulating adaptation to a single irreparable DSB also regulate adaptation to a loss of Cdc13p. For this purpose we created three tlc1Delta cdc13Delta strains also harboring DSB adaptation related mutations tid1Delta, ptc2Delta and rfa1--t11. After deprotection of their telomeres, mutant survivor cells showed similar cell cycle progression patterns as compared to the cells where a single irreparable DSB was introduced. Adaptation defective mutants tid1Delta and ptc2Delta demonstrated an inability to adapt to telomere uncapping and to resume cell cycle. Interestingly, cells harboring the rfa1-t11 allele, which was reported to suppress adaptation defects of other mutations, did not show any distinguishable phenotype in terms of initial adaptation to telomere deprotection; i.e. rfa1-t11 mutant survivors do escape the G2/M arrest and re-enter the cell cycle. However, all three mutant survivor strains failed to produce viable Delta13 capping independent cells, which is consistent with the hypothesis that adaptation to loss of Cdc13p depends on the same pathway as the previously reported adaptation phenomenon.;Finally, we report the surprising finding that if cells had once experienced an adapted Delta13 state, they will re-produce capping negative survivors much more readily. Thus, while a culture of type II survivor cells generates Delta13s at a rate of about 1x10-5 events per division, cells that had been Delta13s and re-transformed with a Cdc13p carrying plasmid will produce capping independent cells at about 1x10-2 events per division. We are currently examining why these cells re-generate Delta13 cell lines more readily and suspect structural differences in telomere terminal sequence arrangements.;In an attempt to identify genes that are involved in the deleterious outcome of an absence of Cdc13p, we screened the yeast gene knock-out library for genes that could suppress the growth defect of cdc13-1 cells at 33°C. For this purpose, we performed an SGA array experiment. We scored for the ability of double mutant haploids to grow at 33°C. Eventually, we hoped to find the elusive genes involved in telomere 5'-end processing (exonucleases).