Mechanism of processive telomerase catalysis revealed by high-resolution optical tweezers

Abstract

Telomerase | Telomere | Optical Tweezers | Single-Molecule Fluorescence | G-quadruplex | Cancer | AgingCorrespondence:schmi706@msu.edu (Twitter: @jenscs83) and mjcomsto@msu.eduTelomere 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 1 . Telomerase is a reverse transcriptase composed of telomerase reverse transcriptase (TERT) 2 , telomerase RNA (TR) 3 , and several accessory proteins 4 . To compensate for telomeric DNA lost during DNA replication, telomerase processively adds GGTTAG repeats to the singlestranded overhangs at chromosome ends by copying the template region within its RNA subunit 5,6 . Between repeat additions, the RNA template must be recycled, which requires disrupting the base-pairing between TR and the substrate DNA 6 . How telomerase remains associated with the substrate DNA during this critical translocation step remains unknown. Here, we demonstrate that stable substrate DNA binding at an anchor site within telomerase facilitates the processive synthesis of telomeric repeats. Using a newly developed single molecule telomerase activity assay utilizing high-resolution optical tweezers, we directly measured stepwise, processive telomerase activity. We found that telomerase tightly associates with its DNA substrate, synthesizing multiple telomeric repeats before releasing them in a single large step. The rate at which product is released from the anchor site closely corresponds to the overall rate of product dissociation from elongating telomerase 7 , suggesting that it is a key parameter controlling telomerase processivity. We observed folding of the released product DNA into G-quadruplex structures. Our results provide detailed mechanistic insights into processive telomerase catalysis, a process critical for telomere length maintenance and therefore cancer cell survival 8,9 .To investigate substrate extension by a single telomerase ribonucleoprotein (RNP), we developed a single molecule telomerase activity assay using dual-trap high-resolution optical tweezers (Fig. 1a). Telomerase and its substrate were attached to separate polystyrene beads (Fig. 1b). The connection between the two beads was formed by the association of telomerase with its substrate DNA ( Fig. 1a,b). When applying a low constant force (4.0-4.5 pN) to the tether, substrate elongation by telomerase was measured as an increase in dis-tance between the two beads ( Fig. 1a). To attach telomerase to the bead, we utilized a 3xFLAG-HaloTag on TERT, modified with biotin ( Fig. 1b,c). This tag did not affect telomerase assembly, catalytic activity, processivity, or stimulation by POT1/TPP1 ( Fig. 1c,d, Extended Data Fig. 1a-c), indicating that it is fully functional.Using this experimental approach, we set out to analyze how telomerase processively synthesizes telomeric repeats. If substrate DNA is only bound to telomerase by basepairing to TR we would expect to observe a sequence of single nucleotide (nt) addition events as a result o...

Structural studies of human telomerase complexes by cryo-electron microscopy

“…Biophysical data shows an anchor site dissociation constant and the Michaelis-Menten constant of telomerase, meaning instead of the hTERC template site, the anchor site is the primary binding site of DNA during telomerase catalysis. While the anchor site does not move, it has been suggested the extended telomere folds back to form a loop and subsequently released (Patrick et al 2019). As mentioned previously, hTPP1-POT1 complexes act as a positive regulator by interacting with telomerase in a coordinating manner to increase telomerase affinity for its substrate, thus increasing the processivity (Figure 1.1.8).…”

“…The current model of telomere elongation is initiated by the recognition and binding of telomeric DNA repeats by the template region of telomerase RNA, TERT and the anchor site (Figure 1.1.7). The 3' end of the telomeric DNA forms a hybrid with the 3' RNA template region, whereas the 5' region of the DNA is postulated to interact with two proposed anchor sites (Greider and Blackburn 1985;Patrick et al 2019;Zaug, Podell, and Cech 2008). Next, the polymerase activity of hTERT extends the 3' telomeric DNA until the 5' boundary of the template region of hTERC.…”

“…While the helix is broken, to prevent the dissociation of the enzyme from the product, another "anchor" site is required. Recently, by biophysical approach experiment, the Schmidt group suggested that the fully assembled human telomerase RNP that is functional at the telomere contains a secondary DNA binding site but does not demonstrate the molecular nature of the binding site (Patrick et al 2019). Other publications suggest the TEN domain contain regions responsible for the anchoring region, due to its spatial proximity to the template binding region when in the catalytic ring formed by TRBD, RT and CTE of TERT.…”