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CST does not evict elongating telomerase but prevents initiation by ssDNA binding

Zaug, Arthur J. and Lim, Ci Ji and Olson, Conner L. and Carilli, Maria T. and Goodrich, Karen J. and Wuttke, Deborah S. and Cech, Thomas R. (2021) CST does not evict elongating telomerase but prevents initiation by ssDNA binding. . (Unpublished)

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The CST complex (CTC1-STN1-TEN1) has been shown to inhibit telomerase extension of the G-strand of telomeres and facilitate the switch to C-strand synthesis by DNA polymerase alpha-primase (pol α-primase). Recently the structure of human CST was solved by cryo-EM, allowing the design of mutant proteins defective in telomeric ssDNA binding and prompting the reexamination of CST inhibition of telomerase. The previous proposal that human CST inhibits telomerase by sequestration of the DNA primer was tested with a series of DNA-binding mutants of CST and modeled by a competitive binding simulation. The DNA-binding mutants had substantially reduced ability to inhibit telomerase, as predicted from their reduced affinity for telomeric DNA. These results provide strong support for the previous primer sequestration model. We then tested whether addition of CST to an ongoing processive telomerase reaction would terminate DNA extension. Pulse-chase telomerase reactions with addition of either wild-type CST or DNA-binding mutants showed that CST has no detectable ability to terminate ongoing telomerase extension in vitro. The same lack of inhibition was observed with or without pol α-primase bound to CST. These results suggest how the switch from telomerase extension to C-strand synthesis may occur.

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Additional Information:The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license. This version posted August 25, 2021. We thank T. Nahreini for her excellent management of the Biochemistry Cell Culture Facility, CU Boulder. T.R.C. is an investigator of the Howard Hughes Medical Institute. Funding: National Institutes of Health [R00 GM131023 to C.L., R01 GM139274 to D.S.W.] and the National Science Foundation [MCB 1716425 to D.S.W.]. M.T.C was supported by the Biological Sciences Initiative funded by the University of Colorado Boulder and a grant from the Howard Hughes Medical Institute through the Science Education Program. Funding for open access charge: Howard Hughes Medical Institute and the University of Colorado Boulder Open Access Fund. Conflict of Interest: T.R.C. is a scientific advisor for Storm Therapeutics and Eikon Therapeutics.
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Howard Hughes Medical Institute (HHMI)UNSPECIFIED
NIHR00 GM131023
NIHR01 GM139274
University of Colorado, BoulderUNSPECIFIED
Record Number:CaltechAUTHORS:20210831-220545152
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Official Citation:CST does not evict elongating telomerase but prevents initiation by ssDNA binding. Arthur J. Zaug, Ci Ji Lim, Conner L. Olson, Maria T. Carilli, Karen J. Goodrich, Deborah S. Wuttke, Thomas R. Cech. bioRxiv 2021.08.25.457677; doi:
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:110674
Deposited By: Tony Diaz
Deposited On:31 Aug 2021 22:14
Last Modified:16 Nov 2021 19:41

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