Real-time detection of human telomerase DNA synthesis by multiplexed single-molecule FRET.
Biophys J
; 122(17): 3447-3457, 2023 09 05.
Article
en En
| MEDLINE
| ID: mdl-37515327
ABSTRACT
Genomic stability in proliferating cells critically depends on telomere maintenance by telomerase reverse transcriptase. Here we report the development and proof-of-concept results of a single-molecule approach to monitor the catalytic activity of human telomerase in real time and with single-nucleotide resolution. Using zero-mode waveguides and multicolor FRET, we recorded the processive addition of multiple telomeric repeats to individual DNA primers. Unlike existing biophysical and biochemical tools, the novel approach enables the quantification of nucleotide-binding kinetics before nucleotide incorporation. Moreover, it provides a means to dissect the unique translocation dynamics that telomerase must undergo after synthesis of each hexameric DNA repeat. We observed an unexpectedly prolonged binding dwell time of dGTP in the enzyme active site at the start of each repeat synthesis cycle, suggesting that telomerase translocation is composed of multiple rate-contributing sub-steps that evade classical biochemical analysis.
Texto completo:
1
Colección:
01-internacional
Banco de datos:
MEDLINE
Asunto principal:
Telomerasa
Tipo de estudio:
Diagnostic_studies
Límite:
Humans
Idioma:
En
Revista:
Biophys J
Año:
2023
Tipo del documento:
Article