The dynamics of yeast telomeres and silencing proteins through the cell cycle

Genes integrated near the telomeres of budding yeast have a variegated patt ern of gene repression that is mediated by the silent information regulator y proteins Sir2p, Sir3p, and Sir4p. Immunolocalization and fluorescence in situ hybridization (FISH) reveal 6-10 perinuclear foci in which silencing p roteins and subtelomeric sequences colocalize, suggesting that these are si tes of Sir-mediated repression. Telomeres lacking subtelomeric repeat eleme nts and the silent mating locus, HML, also localize to the periphery of the nucleus. Conditions that disrupt telomere proximal repression disrupt the focal staining pattern of Sir proteins, but not necessarily the localizatio n of telomeric DNA. To monitor the telomere-associated pools of heterochrom atin-binding proteins (Sir and Rap1 proteins) during mitotic cell division, we have performed immunofluorescence and telomeric FISH on populations of yeast cells synchronously traversing the cell cycle. We observe a partial r elease of Rap1p from telomeres in late G2/M, although telomeres appear to s tay clustered during G2-phase and throughout mitosis. A partial release of Sir3p and Sir4p during mitosis also occurs. This is not observed upon HU ar rest, although other types of DNA damage cause a dramatic relocalization of Sir and Rap1 proteins. The observed cell cycle dynamics were confirmed by direct epifluorescence of a GFP-Rap1p fusion. Using live GFP fluorescence w e show that the diffuse mitotic distribution of GFP-Rap1p is restored to th e interphase pattern of foci in early G1-phase. (C) 2000 Academic Press.