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Real-time detection of human telomerase DNA synthesis by multiplexed single-molecule FRET.
Hentschel, Jendrik; Badstübner, Mareike; Choi, Junhong; Bagshaw, Clive R; Lapointe, Christopher P; Wang, Jinfan; Jansson, Linnea I; Puglisi, Joseph D; Stone, Michael D.
Afiliação
  • Hentschel J; Department of Chemistry and Biochemistry, University of California, Santa Cruz, Santa Cruz, California; Department of Structural Biology, Stanford University School of Medicine, Stanford, California.
  • Badstübner M; Department of Chemistry and Biochemistry, University of California, Santa Cruz, Santa Cruz, California.
  • Choi J; Department of Structural Biology, Stanford University School of Medicine, Stanford, California.
  • Bagshaw CR; Department of Chemistry and Biochemistry, University of California, Santa Cruz, Santa Cruz, California.
  • Lapointe CP; Department of Structural Biology, Stanford University School of Medicine, Stanford, California.
  • Wang J; Department of Structural Biology, Stanford University School of Medicine, Stanford, California.
  • Jansson LI; Department of Chemistry and Biochemistry, University of California, Santa Cruz, Santa Cruz, California.
  • Puglisi JD; Department of Structural Biology, Stanford University School of Medicine, Stanford, California.
  • Stone MD; Department of Chemistry and Biochemistry, University of California, Santa Cruz, Santa Cruz, California. Electronic address: mds@ucsc.edu.
Biophys J ; 122(17): 3447-3457, 2023 09 05.
Article em 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.
Assuntos

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Telomerase Tipo de estudo: Diagnostic_studies Limite: Humans Idioma: En Ano de publicação: 2023 Tipo de documento: Article

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Telomerase Tipo de estudo: Diagnostic_studies Limite: Humans Idioma: En Ano de publicação: 2023 Tipo de documento: Article