Use of non-denaturing Southern hybridization and two dimensional agarose gels to detect putative intermediates in telomere replication inSaccharomyces cerevisiae

Abstract

Telomeres are required for the complete duplication of the ends of linear chromosomes.Saccharomyces telomeres bear ∼350 bps of C_1–3A/TG_1–3 sequences. Previous work using non-denaturing Southern blotting has demonstrated the cell cycle controlled appearance of single stranded TG_1–3 tails on chromosomal and plasmid telomeres (Wellinger et al. submitted). Furthermore it was shown that short linear plasmids carrying an origin of replication derived from 2 μm DNA can circularize at the time of telomere replication (Wellinger et al. submitted). Here we demonstrate that those loci previously shown to acquire single stranded tails are indeed telomeres and that single stranded TG_1–3 cannot be observed in non-telomeric C_1–3A/TG_1–3-tracts. Moreover, we demonstrate that the formation of circular DNA by short linear plasmids is not restricted to plasmids containing a 2 μm origin of replication but can also be detected for plasmids containingARS1.