Sir4 Deficiency Reverses Cell Senescence by Sub-Telomere Recombination.

Telomere shortening results in cellular senescence and the regulatory mechanisms remain unclear. Here, we report that the sub-telomere regions facilitate telomere lengthening by homologous recombination, thereby attenuating senescence in yeast Saccharomyces cerevisiae. The telomere protein complex Sir3/4 represses, whereas Rif1 promotes, the sub-telomere Y' element recombination. Genetic disruption of SIR4 increases Y' element abundance and rescues telomere-shortening-induced senescence in a Rad51-dependent manner, indicating a sub-telomere regulatory switch in regulating organismal senescence by DNA recombination. Inhibition of the sub-telomere recombination requires Sir4 binding to perinuclear protein Mps3 for telomere perinuclear localization and transcriptional repression of the telomeric repeat-containing RNA TERRA. Furthermore, Sir4 repression of Y' element recombination is negatively regulated by Rif1 that mediates senescence-evasion induced by Sir4 deficiency. Thus, our results demonstrate a dual opposing control mechanism of sub-telomeric Y' element recombination by Sir3/4 and Rif1 in the regulation of telomere shortening and cell senescence.

Simultaneous visualisation of the complete sets of telomeres from the MmeI generated terminal restriction fragments in yeasts.

Telomere length is measured using Southern blotting of the chromosomal terminal restriction fragments (TRFs) released by endonuclease digestion in cells from yeast to human. In the budding yeast Saccharomyces cerevisiae, XhoI or PstI is applied to cut the subtelomere Y' element and release TRFs from the 17 subtelomeres. However, telomeres from other 15 X-element-only subtelomeres are omitted from analysis. Here, we report a method for measuring all 32 telomeres in S. cerevisiae using the endonuclease MmeI. Based on analyses of the endonuclease cleavage sites, we found that the TRFs generated by MmeI displayed two distinguishable bands in the sizes of ~500 and ~700 bp comprising telomeres (300 bp) and subtelomeres (200-400 bp). The modified MmeI-restricted TRF (mTRF) method recapitulated telomere shortening and lengthening caused by deficiencies of YKu and Rif1 respectively in S. cerevisiae. Furthermore, we found that mTRF was also applicable to telomere length analysis in S. paradoxus strains. These results demonstrate a useful tool for simultaneous detection of telomeres from all chromosomal ends with both X-element-only and Y'-element subtelomeres in S. cerevisiae species.