De novo telomere addition by Tetrahymena telomerase in vitro.
Previous molecular genetic studies have shown that during programmed chromosomal 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 understood. Here, we show for the first time that telomerase activity is capable in vitro of efficiently elongating completely non-telomeric DNA oligonucleotide primers, consisting of natural telomere-adjacent or random sequences, at low primer concentrations. Telomerase activity isolated from mated or vegetative cells had indistinguishable specificities for nontelomeric and telomeric primers. Consistent with in vivo results, the sequence GGGGT... was the predominant initial DNA sequence added 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 single-stranded non-telomeric primers, primer length up to approximately 30 nucleotides strongly affected the efficiency of telomeric DNA addition. We propose a model for the primer binding site of telomerase for non-telomeric primers to account for these length and structural requirements. We also propose that programmed de novo telomere addition in vivo is achieved through a hitherto undetected intrinsic ability of telomerase to elongate completely non-telomeric sequences.
Wang
H
H
Department of Microbiology and Immunology, University of California San Francisco, 94143, USA.
Blackburn
E H
EH
D000818:Animals; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D008274:Magnesium / Q000494:pharmacology; D019346:Sodium Acetate / Q000494:pharmacology; D013095:Spermidine / Q000494:pharmacology; D013379:Substrate Specificity; D019098:Telomerase / Q000302:isolation & purification / Q000378:metabolism*; D016615:Telomere / Q000378:metabolism; D016808:Tetrahymena thermophila / Q000201:enzymology* / Q000254:growth & development
D017931:DNA Primers; D019346:Sodium Acetate; D019098:Telomerase; D008274:Magnesium; D013095:Spermidine