Abstract
Site-specific recombination in Saccharomyces cerevisiae was used to generate non-replicative DNA rings containing yeast telomeric sequences. In topoisomerase mutants expressing Escherichia coli topoisomerase I, the rings adopted a novel DNA topology consistent with the ability of yeast telomeric DNA to block or retard the axial rotation of DNA. DNA fragments bearing portions of the terminal repeat sequence C1-3 A/TG1-3 were both necessary and sufficient to create a barrier to DNA rotation. Synthetic oligonucleotide sequences containing Rap1p binding sites, a well represented motif in naturally occurring C1-3A arrays, also conferred immobilization; mutant Rap1p binding sites and telomeric sequences from other organisms were not sufficient. DNA anchoring was diminished by addition of competing telomeric sequences, implicating a role for an as yet unidentified limiting trans-acting factor. Though Rap1p is a likely protein constituent of the DNA anchor, deletion of the non-essential C-terminal domain did not affect the topology of telomeric DNA rings. Similarly, disruption of SIR2, SIR3 and SIR4, genes which influence a variety of telomere functions in yeast, also had no effect. We propose that telomeric DNA supports the formation of a SIR-independent macromolecular protein-DNA assembly that hinders the motion of DNA because of its linkage to an insoluble nuclear structure. Potential roles for DNA anchoring in telomere biology are discussed.