Developmentally regulated initiation of DNA synthesis by telomerase: evidence for factor-assisted de novo telomere formation.
Telomerase serves a dual role at telomeres, maintaining tracts of telomere repeats and forming telomeres de novo on broken chromosomes in a process called chromosome healing. In ciliates, both mechanisms are readily observed. Vegetatively growing cells maintain pre-existing telomeres, while cells undergoing macronuclear development fragment their chromosomes and form telomeres de novo. Here we provide the first evidence for developmentally regulated initiation of DNA synthesis by telomerase. In vitro assays were conducted with telomerase from vegetative and developing Euplotes macronuclei using chimeric primers that contained non-telomeric 3' ends and an upstream stretch of telomeric DNA. In developing macronuclei, chimeric primers had two fates: nucleotides were either polymerized directly onto the 3' terminus or residues were removed from the 3' end by endonucleolytic cleavage before polymerization began. In contrast, telomerase from vegetative macronuclei used only the cleavage pathway. Telomere repeat addition onto non-telomeric 3' ends was lost when developing macronuclei were lysed and the contents purified on glycerol gradients. However, when fractions from the glycerol gradient were added back to partially purified telomerase, telomere synthesis was restored. The data indicate that a dissociable chromosome healing factor (CHF) collaborates with telomerase to initiate developmentally programmed de novo telomere formation.
Bednenko
J
J
Department of Biochemistry and Biophysics, Texas A&M University, College Station 77843-2128, USA.
Melek
M
M
Greene
E C
EC
Shippen
D E
DE
D000818:Animals; D000460:Chlorophyta; D018920:Coculture Techniques; D017931:DNA Primers / Q000378:metabolism; D004261:DNA Replication; D016054:DNA, Protozoan / Q000096:biosynthesis*; D016812:Euplotes / Q000235:genetics* / Q000254:growth & development; D009690:Nucleic Acid Conformation; D016053:RNA, Protozoan / Q000378:metabolism; D019098:Telomerase / Q000235:genetics / Q000378:metabolism*; D016615:Telomere / Q000502:physiology*; D013698:Templates, Genetic
D017931:DNA Primers; D016054:DNA, Protozoan; D016053:RNA, Protozoan; D019098:Telomerase