DGTP-DEPENDENT PROCESSIVITY AND POSSIBLE TEMPLATE SWITCHING OF EUPLOTES TELOMERASE

We have measured the processivity of telomeric DNA extension by Euplot es aediculatus telomerase at various concentrations of the nucleotide substrates dGTP and dTTP, The maximum processivity (similar to 3 repea ts) was observed at similar to 100 mu M of each dNTP, Processivity dec reased as the dNTP concentrations were reduced and, surprisingly, as t he concentration of dGTP was increased. Also, the characteristic bandi ng pattern generated by telomerase extension of DNA primers shifted in response to changes in dGTP concentration, One pattern with 8 nt peri odicity was predominant at dGTP concentrations less than or equal to 1 6 mu M, while at greater than or equal to 250 mu M an 8 nt repeat patt ern out-of-phase with the first was observed; at intermediate concentr ations the two patterns coexisted, We propose that two different segme nts of the RNA subunit can serve as the template for repeat synthesis; nt 42-49 at low dGTP concentrations and nt 36-43 at high dGTP concent rations, An alternative model for the low dGTP pattern involves an int ernal pause site but no pause at the end of the template and is, there fore, considered less likely, Because the effects of dGTP on processiv ity and banding pattern appear to be distinct from nucleotide binding in the polymerase active site, we propose a second dGTP binding site i nvolved in template selection and processivity.