Poster abstracts

Poster number 63 submitted by Keontre Hughes

Improved high-resolution optical fleezers methods for investigating human telomerase

Keontre I. Hughes (Michigan State University Physics and Astronomy Department), Ibrahim Elsedek (Michigan State University Physics and Astronomy Department), Kasun B. Senanayake (Michigan State University Physics and Astronomy Department), Jens Schmidt (Michigan State University Department of Obstetrics, Gynecology and Reproductive Biology), Matthew J. Comstock (Michigan State University Physics and Astronomy Department)

Abstract:
Telomere maintenance by telomerase is critical for continuous proliferation of human cells and is 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. Using a single-molecule telomerase activity assay utilizing high-resolution optical tweezers, our collaboration recently demonstrated in our (Patrick et al., Nature Chem Bio, 2020) that substrate DNA binding at an anchor site within telomerase facilitates the processive synthesis of telomeric repeats. The DNA synthesized by telomerase can then be recaptured by the anchor site or fold/unfold into G-quadruplex structures. To further investigate the mechanisms guiding telomerase catalysis, anchor site release/recapture, and related intra-molecular interfaces (e.g. telomerase RNA folding in relation to associated proteins such as histone H2A/H2B dimers), we present numerous enhancements to our previous methodology both in tether construction and tweezers methods. The new tweezers methods address the need to assay human telomerase at low force, with high sensitivity over long elongation distances and in part address previous errors introduced acousto optic device (AOD) beam steering of time-shared dual-trap optical tweezers. We also present preliminary force spectroscopy of human telomerase RNA CR4/5 region, which associates with the histone H2A/H2B dimer.

References:
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

Keywords: Telomerase , Optical Tweezers