Euplotes telomerase: evidence for limited base-pairing during primer elongation and dGTP as an effector of translocation.
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 (koff), 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 koff 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 koff 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 <10 base pairs, with the remaining energy contributed by other parts of telomerase. Most primers exhibited biphasic dissociation kinetics, with variations in both the amount in each phase and the rate for each phase. Since the cross-links monitored in the dissociation assay were all formed with the 5' region of the primer, the two phases may arise from different base-pairing registers with the RNA template, possibly representing pre- and post-translocation complexes. A shift from slow phase to fast phase dissociation was observed in the presence of dGTP, which may implicate dGTP as a positive effector of translocation.
Hammond
P W
PW
Department of Chemistry and Biochemistry, Howard Hughes Medical Institute, University of Colorado, Boulder, Colorado 80309-0215, USA.
Cech
T R
TR
D000818:Animals; D003432:Cross-Linking Reagents; D017931:DNA Primers / Q000378:metabolism*; D003848:Deoxyguanine Nucleotides / Q000378:metabolism; D016812:Euplotes / Q000201:enzymology*; D008957:Models, Genetic; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D012313:RNA / Q000378:metabolism*; D012261:Ribonucleoproteins / Q000378:metabolism*; D019098:Telomerase / Q000378:metabolism*; D016615:Telomere / Q000378:metabolism
D003432:Cross-Linking Reagents; D017931:DNA Primers; D003848:Deoxyguanine Nucleotides; D012261:Ribonucleoproteins; D012313:RNA; C029603:deoxyguanosine triphosphate; D019098:Telomerase