The coherence of synthetic telomeres.

The chromosomal telomeres of Oxytricha were synthesized and their ability to cohere examined on non-denaturing acrylamide gels containing the stabilizing cation K+. At least 5 different mobility species were observed, in addition to that of the monomeric telomere. By cohering synthetic telomeres containing different lengths of subtelomeric DNA, we showed that each of the different mobility species was a dimer of two telomeres. Since the different mobility species did not differ in numbers or sequences of nucleotides, they must correspond to different molecular shapes probably caused by different degrees of bending of the dimer. Paradoxically, telomeres with longer subtelomeric stems cohered more efficiently. In the presence of K+, solutions had to be heated to over 90 degrees before the telomeres separated. Various synthetic constructs, restriction endonuclease and dimethyl sulfate protection experiments showed that the only nucleotides involved in the cohered structures were the 16 base 'tails' of sequence 3'G4T4G4T4. Extension of this motif was actually inimical to coherence. Oligomers containing 2 G4T4 motifs protected their GN7 positions by forming dimers, those with 5 G4T4 could do so by internal folding, but the 3' terminal group of G4 was left unprotected. This suggests that only four groups of G4 are necessary for the cohered structure. Single-chain specific nuclease, S1, as well as osmium tetroxide, which oxidizes the thymine residues of single chains, reacted less efficiently with the cohered structures. Synthetic telomeres containing inosine replacing guanosine were not observed to cohere, indicating that the C2-NH2 is strongly stabilizing. The cohered structures appear to be unusually compact and sturdy units in which four G4 blocks form quadruplexes stabilized by K+. A new model for the cohered structure is presented.