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

Telomeres are required for the complete duplication of the ends of linear chromosomes. Saccharomyces telomeres bear approximately 350 bps of C1-3A/TG1-3 sequences. Previous work using non-denaturing Southern blotting has demonstrated the cell cycle controlled appearance of single stranded TG1-3 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 microns 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 TG1-3 cannot be observed in non-telomeric C1-3A/TG1-3-tracts. Moreover, we demonstrate that the formation of circular DNA by short linear plasmids is not restricted to plasmids containing a 2 microns origin of replication but can also be detected for plasmids containing ARS1.