Telomere DNA G-quadruplex folding within actively extending human telomerase

Abstract

Telomerase maintains telomere length by reverse transcribing short G-rich DNA repeat sequences from its internal RNA template. G-rich telomere DNA repeats readily fold into Gquadruplex (GQ) structures in vitro, and the presence of GQ-prone sequences throughout the genome introduces challenges to replication in vivo. Using a combination of ensemble and single-molecule telomerase assays we discovered that GQ folding of the nascent DNA product during processive addition of multiple telomere repeats modulates the kinetics of telomerase catalysis and dissociation. Telomerase reactions performed with telomere DNA primers of varying sequence or using K + versus Li + salts yield changes in DNA product profiles consistent with formation of GQ structure within the telomerase-DNA complex. Single-molecule FRET experiments reveal complex DNA structural dynamics during real-time catalysis, supporting the notion of nascent product folding within the active telomerase complex. To explain the observed distributions of telomere products, we fit telomerase time series data to a global kinetic model that converges to a unique set of rate constants describing each successive telomere repeat addition cycle. Our results highlight the potential influence of the intrinsic folding properties of telomere DNA during telomerase catalysis and provide a detailed characterization of GQ modulation of polymerase function. SIGNIFICANCETelomeres protect the ends of linear chromosomes from illicit DNA processing events that can threaten genome stability. Telomere structure is built upon repetitive G-rich DNA repeat sequences that have the ability to fold into stable secondary structures called G-quadruplexes (GQs). In rapidly dividing cells, including the majority of human cancers, telomeres are maintained by the specialized telomerase enzyme. Thus, telomerase and its telomere DNA substrates represent important targets for developing novel cancer drugs. In this work, we provide evidence for GQ folding within the newly synthesized DNA product of an actively extending telomerase enzyme. Our results highlight the delicate interplay between the structural properties of telomere DNA and telomerase function.

Observation of processive telomerase catalysis using high-resolution optical tweezers

“…We next sought to determine the molecular mechanisms that underlie the reverse steps observed in telomerase catalysis trajectories. Reverse steps are most likely the consequence of two distinct processes: 1) Rebinding of product DNA to the anchor site, or 2) folding of product into a G-quadruplex (GQ) structure, which has been shown to affect telomerase catalysis 4 . In extended telomerase tethers, the most common reverse and forward step size is approximately 15 nt (25% and 15% of transitions, respectively, Fig.…”

“…A key challenge for telomere maintenance is the G-rich nature of the telomeric repeats in human cells. Specifically, the formation of G-quadruplex (GQ) structures has been shown to affect the replication of telomeres 3 and telomerase catalysis 4 . GQs are nucleic acid structures that are formed by four single stranded telomeric repeats 5 , 6 .…”

Genome-Wide Discovery of G-Quadruplexes in Wheat: Distribution and Putative Functional Roles

“…Our results show that G4 motifs are distributed throughout the chromosome; yet, are enriched mostly at chromosome ends. The chromosome ends containing telomeres are well-known regions for G4 structure formation (Neidle 2010;Jansson et al 2019;Wu et al 2020). Other than chromosomal end points, some of the G4 motif enriched regions were the regions enriched for high confidence gene models, such as ~500-600 Mb regions on the chromosomes 4A (coordinate for the first peak: 605,306,311 bp), 5A (516,960,980 bp), and 7D (542,590,594 bp) and ~100 Mb regions on the chromosomes 6B (123,972,511 bp) and 7A (75,769,595 bp).…”

Folding heterogeneity in the essential human telomerase RNA three-way junction

“…In vitro reconstitution of human telomerase Human telomerase was reconstituted in rabbit reticulocyte lysate (RRL) using the TNT Quick Coupled Transcription/Translation system (Promega) as described previously (Weinrich et al 1997;Jansson et al 2019). In Lo-bind tubes (Eppendorf), 200 µl of TnT quick mix was combined with 5 µg of pNFLAG-hTERT plasmid as well as 1 µM of in vitro transcribed and unlabeled hTR t/PK and CR4/5 fragments.…”