2 research outputs found
Recommended from our members
Telomere DNA G-quadruplex folding within actively extending human telomerase
Telomerase reverse transcribes short guanine (G)-rich DNA repeat sequences from its internal RNA template to maintain telomere length. G-rich telomere DNA repeats readily fold into G-quadruplex (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 GQ-stabilizing K+ versus GQ-destabilizing Li+ salts yielded changes in DNA product profiles consistent with formation of GQ structures within the telomerase-DNA complex. Addition of the telomerase processivity factor POT1-TPP1 altered the DNA product profile, but was not sufficient to recover full activity in the presence of Li+ cations. This result suggests GQ folding synergizes with POT1-TPP1 to support telomerase function. Single-molecule Förster resonance energy transfer experiments reveal complex DNA structural dynamics during real-time catalysis in the presence of K+ but not Li+, supporting the notion of nascent product folding within the active telomerase complex. To explain the observed distributions of telomere products, we globally fit telomerase time-series data to a kinetic model that converges to a 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
A method for campus-wide SARS-CoV-2 surveillance at a large public university
The systematic screening of asymptomatic and pre-symptomatic individuals is a powerful tool for controlling community transmission of infectious disease on college campuses. Faced with a paucity of testing in the beginning of the COVID-19 pandemic, many universities developed molecular diagnostic laboratories focused on SARS-CoV-2 diagnostic testing on campus and in their broader communities. We established the UC Santa Cruz Molecular Diagnostic Lab in early April 2020 and began testing clinical samples just five weeks later. Using a clinically-validated laboratory developed test (LDT) that avoided supply chain constraints, an automated sample pooling and processing workflow, and a custom laboratory information management system (LIMS), we expanded testing from a handful of clinical samples per day to thousands per day with the testing capacity to screen our entire campus population twice per week. In this report we describe the technical, logistical, and regulatory processes that enabled our pop-up lab to scale testing and reporting capacity to thousands of tests per day