Reiterative dG addition by Euplotes crassus telomerase during extension of non-telomeric DNA.
Telomerase from the ciliate Euplotes crassus incorporates G4T4telomeric repeats onto both telomeric and non-telomeric single-stranded DNA 3'-ends via reverse transcription of a templating domain in its RNA subunit. Here we describe an unusual mode of template copying that is characteristic of DNA synthesis onto non-telomeric 3'-ends in vitro . When dTTP was eliminated from telomerase reactions, telomeric primers or DNA products generated from the telomerase endonuclease were extended by precise copying of the RNA template. In contrast, telomerase catalyzed the addition of up to 13 dG residues onto primers with non-telomeric 3'-ends under the same reaction conditions. Introducing mismatches in the 3'-terminus of telomeric primers that reduced primer complementarity to the RNA template induced reiterative dG incorporation, indicating that the reaction is influenced by Watson-Crick base pair formation between the primer and the RNA template. Unexpectedly, the reiterative dG addition mode was confined to telomerase derived from developing cells that undergo new telomere formation. This reaction was not observed in vegetatively growing cells. We postulate that indiscriminate dG addition by telomerase occurs by reiterative copying of C residues in the telomerase RNA templating domain and reflects lateral instability of the primer-template interaction during de novo telomere formation.
Bednenko
J
J
Department of Biochemistry and Biophysics, Texas A&M University, College Station, TX 77843-2128, USA.
Melek
M
M
Shippen
D E
DE
D000818:Animals; D002453:Cell Cycle; D002467:Cell Nucleus / Q000201:enzymology; D017931:DNA Primers; D016054:DNA, Protozoan / Q000096:biosynthesis*; D016812:Euplotes / Q000201:enzymology / Q000235:genetics*; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D011068:Poly G / Q000096:biosynthesis*; D013379:Substrate Specificity; D019098:Telomerase / Q000378:metabolism*; D016615:Telomere / Q000378:metabolism*; D014158:Transcription, Genetic
D017931:DNA Primers; D016054:DNA, Protozoan; D009838:Oligodeoxyribonucleotides; D011068:Poly G; C026628:poly(dG); D019098:Telomerase