Yeast telomeres: how to ignore essential double-strand DNA breaks?

Abstract

DNA looping is one of the mechanisms involved in telomere maintenance. It probably provides a solution not only to ‘the end-replication problem’, but also for the protection of chromosomal ends against degradation enzymes and, as typical double-strand breaks, from DNA repair machinery. Telomeric loops (t-loops) formed by an invasion of protruding 3’ overhangs into the double- stranded telomeric regions were observed in a variety of organisms ranging from ciliates to mammals. Genetic data indicate that looping also occurs at the telomeres of Saccharomyces cerevisiae, suggesting its importance for telomere function in yeast. However, several observations argue against the presence of ‘true’ t-loops in the budding yeast telomeres (e.g. the lack of TRF- like protein, heterogeneous telomeric sequences). Instead, telomeres in S. cerevisiae appear to form fold-back structures mediated by protein-protein interactions. To directly visualize the telomeric structure in budding yeast, we developed a system based on a mini-chromosome carrying an array of lac operator sequences allowing its purification by the lac repressor affinity column. In contrast to budding yeast, the fission yeast Schizosaccharomyces pombe contains a homologue of the human telomeric protein TRF2, designated Taz1p. As the TRF2 protein has been implicated in remodelling telomeres into t-loops, the ability of Taz1p to promote t-loop formation is examined by electron microscopy using purified protein and synthetic templates containing a double-stranded fission yeast telomeric tract. Our studies should shed some light not only on telomeric architecture in yeast, but should also be instrumental in deciphering detailed telomeric structure in higher eukaryotes.