Euplotes Telomerase:  Evidence for Limited Base-Pairing during Primer Elongation and dGTP as an Effector of Translocation

• Published in: Biochemistry (Easton) 1998-04, Vol.37 (15), p.5162-5172
• Main Authors: Hammond, Philip W, Cech, Thomas R
• Format: Article
• Language: English
• Subjects: Animals; Cross-Linking Reagents; Deoxyguanine Nucleotides - metabolism; DNA Primers - metabolism; Euplotes - enzymology; Models, Genetic; Nucleic Acid Conformation; Protein Binding; Ribonucleoproteins - metabolism; RNA - metabolism; Telomerase - metabolism; Telomere - metabolism
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi972988o

The telomeric sequence repeats at the ends of eukaryotic chromosomes are maintained by the ribonucleoprotein enzyme telomerase. Telomeric DNA primers are bound by telomerase both at the active site, which includes base-pairing with the RNA template, and at a second anchor site. The stabilities of Euplotes aediculatus primer−telomerase complexes were determined by measuring their dissociation rates (k off), using an assay involving photo-cross-linking at the anchor site. The primer length was varied, and mismatched substitutions were introduced in a systematic manner. We observed that k off does not scale with primer length as expected for accumulated primer-template base-pairing. This suggests that telomerase maintains a more-or-less constant number of base pairs, similar to the transcription bubble maintained by RNA polymerase. An upper limit was estimated by comparing the experimental k off for the primer−telomerase complex to that of a model DNA−RNA duplex. All the binding energy could be attributed to 10 or 11 base pairs; alternatively, there could be