Multiple mechanisms for elongation processivity within the reconstituted tetrahymena telomerase holoenzyme.
To maintain telomeres, telomerase evolved a unique biochemical activity: the use of a single-stranded RNA template for the synthesis of single-stranded DNA repeats. High repeat addition processivity (RAP) of the Tetrahymena telomerase holoenzyme requires association of the catalytic core with the telomere adaptor subcomplex (TASC) and an RPA1-related subunit (p82 or Teb1). Here, we used DNA binding and holoenzyme reconstitution assays to investigate the mechanism by which Teb1 and TASC confer high RAP. We show that TASC association with the recombinant telomerase catalytic core increases enzyme activity. Subsequent association of the Teb1 C-terminal domain with TASC confers the capacity for high RAP even though the Teb1 C-terminal domain does not provide a high-affinity DNA interaction site. Efficient RAP also requires suppression of nascent product folding mediated by the central Teb1 DNA-binding domains (DBDs). These sequence-specific high-affinity DBDs of Teb1 can be functionally substituted by the analogous DBDs of Tetrahymena Rpa1 to suppress nascent product folding but only if the Rpa1 high-affinity DBDs are physically tethered into holoenzyme context though the Teb1 C-terminal domain. Overall, our findings reveal multiple mechanisms and multiple surfaces of protein-DNA and protein-protein interaction that give rise to elongation processivity in the synthesis of a single-stranded nucleic acid product.
Min
Bosun
B
Department of Molecular and Cell Biology, University of California, Berkeley, California 94720-3200, USA.
Collins
Kathleen
K
D002384:Catalysis; D004247:DNA / Q000737:chemistry; D004277:DNA, Single-Stranded / Q000737:chemistry; D020035:Holoenzymes / Q000737:chemistry*; D008954:Models, Biological; D009154:Mutation; D009690:Nucleic Acid Conformation; D009696:Nucleic Acids / Q000737:chemistry; D011485:Protein Binding; D017510:Protein Folding; D025941:Protein Interaction Mapping; D017434:Protein Structure, Tertiary; D019098:Telomerase / Q000737:chemistry*; D013768:Tetrahymena / Q000201:enzymology*
D004277:DNA, Single-Stranded; D020035:Holoenzymes; D009696:Nucleic Acids; D004247:DNA; D019098:Telomerase