DE NOVA TELOMERE ADDITION BY TETRAHYMENA TELOMERASE IN-VITRO

Previous molecular genetic studies have shown that during programmed c hromosomal healing, telomerase adds telomeric repeats directly to non- telomeric sequences in Tetrahymena, forming de novo telomeres. However , the biochemical mechanism underlying this process is not well unders tood. Here, we show for the first time that telomerase activity is cap able in vitro of efficiently elongating completely non-telomeric DNA o ligonucleotide primers, consisting of natural telomere-adjacent or ran dom sequences, at low primer concentrations, Telomerase activity isola ted from mated or vegetative cells had indistinguishable specificities for nontelomeric and telomeric primers, Consistent with in vivo resul ts, the sequence GGGGT... was the predominant initial DNA sequence add ed by telomerase in vitro onto the 3' end of the non-telomeric primers . The 3' and 5' sequences of the primer both influenced the efficiency and pattern of de novo telomeric DNA addition, Priming of telomerase by double-stranded primers with overhangs of various lengths showed a requirement for a minimal 3' overhang of 20 nucleotides. With fully si ngle-stranded non-telomeric primers, primer length up to similar to 30 nucleotides strongly affected the efficiency of telomeric DNA additio n, We propose a model for the primer binding site of telomerase for no n-telomeric primers to account for these length and structural require ments, We also propose that programmed de novo telomere addition in vi vo is achieved through a hitherto undetected intrinsic ability of telo merase to elongate completely non-telomeric sequences.