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Observation of processive telomerase catalysis using high-resolution optical tweezers

Abstract

Telomere maintenance by telomerase is essential for continuous proliferation of human cells and is vital for the survival of stem cells and 90% of cancer cells. To compensate for telomeric DNA lost during DNA replication, telomerase processively adds GGTTAG repeats to chromosome ends by copying the template region within its RNA subunit. Between repeat additions, the RNA template must be recycled. How telomerase remains associated with substrate DNA during this critical translocation step remains unknown. Using a single-molecule telomerase activity assay utilizing high-resolution optical tweezers, we demonstrate that stable substrate DNA binding at an anchor site within telomerase facilitates the processive synthesis of telomeric repeats. The product DNA synthesized by telomerase can be recaptured by the anchor site or fold into G-quadruplex structures. Our results provide detailed mechanistic insights into telomerase catalysis, a process of critical importance in aging and cancer.

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Fig. 1: A single-molecule telomerase extension assay using high-resolution dual-trap optical tweezers.
Fig. 2: Telomerase releases multiple telomeric repeats in a single step.
Fig. 3: Analysis of processive DNA synthesis and product release by telomerase.
Fig. 4: Analysis of telomerase catalysis in the presence of LiCl.
Fig. 5: Dissociation from the anchor site controls product release by telomerase.
Fig. 6: Model for processive telomerase catalysis.

Data availability

All data generated or analyzed during this study are included in this manuscript (and its Supplementary Information). Reagents and raw data are available from J.C.S. and M.J.C. upon request.

Code availability

Custom MATLAB code used to analyze the data generated in this study is available from M.J.C. upon request.

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Acknowledgements

We thank J. Nandakumar and I. Cheeseman for comments on the manuscript. This work was support by grants from the NSF (grant no. MCB-1919439) to M.J.C. and the NIH (grant no. R00 GM120386) to J.C.S. J.C.S. is a Damon Runyon Dale F. Frey Scientist supported (in part) by the Damon Runyon Cancer Research Foundation (grant no. DFS-24-17).

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Contributions

Experiments were designed by E.M.P., M.J.C. and J.C.S. Optical trapping with telomerase was carried out by E.M.P. Optical trapping with the multi-G-quadruplex construct was carried out by E.M.P. Optical trapping with the single-G-quadruplex construct was carried out by J.S. and B.P. All other sample preparations and experiments were carried out by E.M.P. and J.C.S. E.M.P., M.J.C. and J.C.S. analyzed data and wrote the manuscript.

Corresponding authors

Correspondence to Matthew J. Comstock or Jens C. Schmidt.

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Supplementary Figs. 1–5 and Table 1.

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Patrick, E.M., Slivka, J.D., Payne, B. et al. Observation of processive telomerase catalysis using high-resolution optical tweezers. Nat Chem Biol 16, 801–809 (2020). https://doi.org/10.1038/s41589-020-0478-0

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