YEAST TELOMERIC SEQUENCES FUNCTION AS CHROMOSOMAL ANCHORAGE POINTS IN-VIVO

Site-specific recombination in Saccharomyces cerevisiae was used to ge nerate 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 C-1-3 A/TG (1-3) were both necessary and sufficient to create a barrier to DNA ro tation, Synthetic oligonucleotide sequences containing Rap1p binding s ites, a well represented motif in naturally occurring C(1-3)A arrays, also conferred immobilization; mutant Rap1p binding sites and telomeri c sequences from other organisms were not sufficient, DNA anchoring wa s diminished by addition of competing telomeric sequences, implicating a role for an as yet unidentified limiting trans-acting factor, Thoug h Rap1p is a likely protein constituent of the DNA anchor, deletion of the non-essential C-terminal domain did not affect the topology of te lomeric 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 moti on of DNA because of its linkage to an insoluble nuclear structure, Po tential roles for DNA anchoring in telomere biology are discussed.