POT1–TPP1 enhances telomerase processivity by slowing primer dissociation and aiding translocation

Abstract

Telomerase contributes to chromosome end replication by synthesizing repeats of telomeric DNA, and the telomeric DNA-binding proteins protection of telomeres (POT1) and TPP1 synergistically increase its repeat addition processivity. To understand the mechanism of increased processivity, we measured the effect of POT1-TPP1 on individual steps in the telomerase reaction cycle. Under conditions where telomerase was actively synthesizing DNA, POT1-TPP1 bound to the primer decreased primer dissociation rate. In addition, POT1-TPP1 increased the translocation efficiency. A template-mutant telomerase that synthesizes DNA that cannot be bound by POT1-TPP1 exhibited increased processivity only when the primer contained at least one POT1-TPP1-binding site, so a single POT1-TPP1-DNA interaction is necessary and sufficient for stimulating processivity. The POT1-TPP1 effect is specific, as another single-stranded DNA-binding protein, gp32, cannot substitute. POT1-TPP1 increased processivity even when substoichiometric relative to the DNA, providing evidence for a recruitment function. These results support a model in which POT1-TPP1 enhances telomerase processivity in a manner markedly different from the sliding clamps used by DNA polymerases.

Expression of hPOT1 in HeLa Cells and the Probability of Gene Variation of hpot1 Exon14 in Endometrial Cancer are Much Higher than in Other Cancers

“…The procedures were performed according to previous publication (Latrick et al, 2010). In brief, HeLa cells nuclear extracts were prepared according to anterior described method Nuclear Protein Extraction for identifying protein-DNA interaction assay.…”

Replication of Telomeres and the Regulation of Telomerase

“…The single-stranded telomere-binding protein Pot1 is an inhibitor of telomerase per se when binding to the 3 0 end of the substrate (Kelleher et al 2005;Lei et al 2005). When associated with Tpp1, however, Pot1 -Tpp1 decreases the dissociation of primer from telomerase and increases the efficiency of translocation (Wang et al 2007;Latrick and Cech 2010). Therefore, the Pot1 -Tpp1 complex increases repeat addition processivity of human telomerase severalfold.…”

Evolution of CST function in telomere maintenance

“…TPP1 also interacts with telomerase 51,52 and enhances its action by increasing enzyme processivity. 53 Thus, TPP1 could potentially function in a manner similar to Cdc13 by serving to first enhance telomerase activity and then to recruit DNA Polα/primase via interactions with CST.…”