EUPLOTES TELOMERASE - EVIDENCE FOR LIMITED BASE-PAIRING DURING PRIMERELONGATION AND DGTP AS AN EFFECTOR OF TRANSLOCATION

The telomeric sequence repeats at the ends of eukaryotic chromosomes a re maintained by the ribonucleoprotein enzyme telomerase. Telomeric DN A primers are bound by telomerase both at the active site, which inclu des base-pairing with the RNA template, and at a second anchor site. T he stabilities of Euplotes aediculatus primer-telomerase complexes wer e determined by measuring their dissociation rates (k(off)), using an assay involving photo-cross-linking at the anchor site. The primer len gth was varied, and mismatched substitutions were introduced in a syst ematic manner. We observed that k(off) does not scale with primer leng th as expected for accumulated primer-template base-pairing. This sugg ests that telomerase maintains a more-or-less constant number of base pairs, similar to the transcription bubble maintained by RNA polymeras e. An upper limit was estimated by comparing the experimental k(off) f or the primer-telomerase complex to that of a model DNA-RNA duplex. Al l the binding energy could be attributed to 10 or 11 base pairs; alter natively, there could be <10 base pairs, with the remaining energy con tributed by other parts of telomerase. Most primers exhibited biphasic dissociation kinetics, with variations in both the amount in each pha se 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 th e RNA template, possibly representing pre-and post-translocation compl exes. A shift from slow phase to fast phase dissociation was observed in the presence of dGTP, which may implicate dGTP as a positive effect or of translocation.