Requirements for the dGTP-dependent repeat addition processivity of recombinant Tetrahymena telomerase

Telomerase is a reverse transcriptase responsible for adding simple sequenc e repeats to chromosome 3'-ends. The template for telomeric repeat synthesi s is carried within the RNA component of the telomerase ribonucleoprotein c omplex. Telomerases can copy their internal templates with repeat addition processivity, reusing the same template multiple times in the extension of a single primer. For some telomerases, optimal repeat addition processivity requires high micromolar dGTP concentrations, a much higher dGTP concentra tion than required for processive nucleotide addition within a repeat. We h ave investigated the requirements for dGTP-dependent repeat addition proces sivity using recombinant Tetrahymena telomerase. By altering the template s equence, we show that repeat addition processivity retains the same dGTP-de pendence even if dGTP is not the first nucleotide incorporated in the secon d repeat. Furthermore, no dNTP other than dGTP can stimulate repeat additio n processivity, even if it is the first nucleotide incorporated in the seco nd repeat. Using structural variants of dGTP, we demonstrate that the stimu lation of repeat addition processivity is specific for dGTP base and sugar constituents but requires only a single phosphate group, However, all nucle otides that stimulate repeat addition processivity also inhibit or compete with dGTP incorporation into product DNA, By assaying telomerase complexes reconstituted with a variety of altered templates, we find that repeat addi tion processivity has an unanticipated template or product sequence specifi city. Finally, we show that a novel, nascent product DNA binding site estab lishes dGTP-dependent repeat addition processivity.