Telomerase contributes to telomere replication by synthesizing repeats of DNA, and two telomeric DNA-binding proteins, POT1 and TPP1, synergistically increase its repeat addition processivity. To study the mechanism of increased processivity, I determined that POT1-TPP1 does not decrease the dissociation rate of telomerase from DNA, prompting reconsideration of the previously proposed sliding and stationary clamp models. Instead, POT1-TPP1 increases the translocation rate and efficiency. A template-mutant telomerase, which synthesizes DNA that cannot be bound by POT1-TPP1, exhibits increased processivity only when at least one POT1-TPP1 binding site is present, evidence for a required POT1-TPP1-DNA interaction during elongation. The effects of POT1-TPP1 are specific as another single-stranded DNA binding protein, gp32, decreases telomerase processivity. POT1-TPP1 stimulates processivity even when substoichiometric relative to the DNA, providing evidence for a recruitment function. I propose a model where POT1-TPP1 must be displaced from the end of a telomere in order for telomerase to initiate, and then, during extension, POT1-TPP1 binds to a nascent telomeric DNA repeat to facilitate the translocation step of repeat addition processivity.
|Advisor:||Cech, Thomas R.|
|Commitee:||Batey, Robert T., McHenry, Charles S., Pace, Norman R., Wuttke, Deborah S.|
|School:||University of Colorado at Boulder|
|School Location:||United States -- Colorado|
|Source:||DAI-B 70/07, Dissertation Abstracts International|
|Keywords:||POT1, Processivity, TPP1, Telomerase|
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