Requirements for the dGTP-dependent repeat addition processivity of recombinant Tetrahymena telomerase.
Telomerase is a reverse transcriptase responsible for adding simple sequence repeats to chromosome 3'-ends. The template for telomeric repeat synthesis is carried within the RNA component of the telomerase ribonucleoprotein complex. 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 concentration than required for processive nucleotide addition within a repeat. We have investigated the requirements for dGTP-dependent repeat addition processivity using recombinant Tetrahymena telomerase. By altering the template sequence, we show that repeat addition processivity retains the same dGTP-dependence even if dGTP is not the first nucleotide incorporated in the second repeat. Furthermore, no dNTP other than dGTP can stimulate repeat addition processivity, even if it is the first nucleotide incorporated in the second repeat. Using structural variants of dGTP, we demonstrate that the stimulation of repeat addition processivity is specific for dGTP base and sugar constituents but requires only a single phosphate group. However, all nucleotides 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 addition processivity has an unanticipated template or product sequence specificity. Finally, we show that a novel, nascent product DNA binding site establishes dGTP-dependent repeat addition processivity.
Hardy
C D
CD
Division of Biochemistry and Molecular Biology, Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, CA 94720-3204, USA.
Schultz
C S
CS
Collins
K
K
D000818:Animals; D001483:Base Sequence; D004247:DNA / Q000378:metabolism; D003848:Deoxyguanine Nucleotides / Q000378:metabolism*; D008957:Models, Genetic; D015800:Protozoan Proteins / Q000378:metabolism; D011994:Recombinant Proteins; D019098:Telomerase / Q000378:metabolism*; D013698:Templates, Genetic; D013768:Tetrahymena / Q000201:enzymology*
D003848:Deoxyguanine Nucleotides; D015800:Protozoan Proteins; D011994:Recombinant Proteins; C029603:deoxyguanosine triphosphate; D004247:DNA; D019098:Telomerase