POT1-TPP1 enhances telomerase processivity by slowing primer dissociation and aiding translocation.
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.
Latrick
Chrysa M
CM
Department of Chemistry and Biochemistry, Howard Hughes Medical Institute, University of Colorado-Boulder, 80309-0215, USA.
Cech
Thomas R
TR
D000818:Animals; D001692:Biological Transport / Q000235:genetics / Q000502:physiology; D002460:Cell Line; D017931:DNA Primers / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D006801:Humans; D008954:Models, Biological; D011817:Rabbits; D000089804:Shelterin Complex; D019098:Telomerase / Q000235:genetics / Q000378:metabolism*; D034501:Telomere-Binding Proteins / Q000378:metabolism
C495876:ACD protein, human; D017931:DNA Primers; D004268:DNA-Binding Proteins; C440849:POT1 protein, human; D000089804:Shelterin Complex; D034501:Telomere-Binding Proteins; D019098:Telomerase