Cargando…

Single-molecule Taq DNA polymerase dynamics

Taq DNA polymerase functions at elevated temperatures with fast conformational dynamics—regimes previously inaccessible to mechanistic, single-molecule studies. Here, single-walled carbon nanotube transistors recorded the motions of Taq molecules processing matched or mismatched template–deoxynucleo...

Descripción completa

Detalles Bibliográficos
Autores principales: Turvey, Mackenzie W., Gabriel, Kristin N., Lee, Wonbae, Taulbee, Jeffrey J., Kim, Joshua K., Chen, Silu, Lau, Calvin J., Kattan, Rebecca E., Pham, Jenifer T., Majumdar, Sudipta, Garcia, Davil, Weiss, Gregory A., Collins, Philip G.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Association for the Advancement of Science 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8916733/
https://www.ncbi.nlm.nih.gov/pubmed/35275726
http://dx.doi.org/10.1126/sciadv.abl3522
_version_ 1784668385197424640
author Turvey, Mackenzie W.
Gabriel, Kristin N.
Lee, Wonbae
Taulbee, Jeffrey J.
Kim, Joshua K.
Chen, Silu
Lau, Calvin J.
Kattan, Rebecca E.
Pham, Jenifer T.
Majumdar, Sudipta
Garcia, Davil
Weiss, Gregory A.
Collins, Philip G.
author_facet Turvey, Mackenzie W.
Gabriel, Kristin N.
Lee, Wonbae
Taulbee, Jeffrey J.
Kim, Joshua K.
Chen, Silu
Lau, Calvin J.
Kattan, Rebecca E.
Pham, Jenifer T.
Majumdar, Sudipta
Garcia, Davil
Weiss, Gregory A.
Collins, Philip G.
author_sort Turvey, Mackenzie W.
collection PubMed
description Taq DNA polymerase functions at elevated temperatures with fast conformational dynamics—regimes previously inaccessible to mechanistic, single-molecule studies. Here, single-walled carbon nanotube transistors recorded the motions of Taq molecules processing matched or mismatched template–deoxynucleotide triphosphate pairs from 22° to 85°C. By using four enzyme orientations, the whole-enzyme closures of nucleotide incorporations were distinguished from more rapid, 20-μs closures of Taq’s fingers domain testing complementarity and orientation. On average, one transient closure was observed for every nucleotide binding event; even complementary substrate pairs averaged five transient closures between each catalytic incorporation at 72°C. The rate and duration of the transient closures and the catalytic events had almost no temperature dependence, leaving all of Taq’s temperature sensitivity to its rate-determining open state.
format Online
Article
Text
id pubmed-8916733
institution National Center for Biotechnology Information
language English
publishDate 2022
publisher American Association for the Advancement of Science
record_format MEDLINE/PubMed
spelling pubmed-89167332022-03-21 Single-molecule Taq DNA polymerase dynamics Turvey, Mackenzie W. Gabriel, Kristin N. Lee, Wonbae Taulbee, Jeffrey J. Kim, Joshua K. Chen, Silu Lau, Calvin J. Kattan, Rebecca E. Pham, Jenifer T. Majumdar, Sudipta Garcia, Davil Weiss, Gregory A. Collins, Philip G. Sci Adv Physical and Materials Sciences Taq DNA polymerase functions at elevated temperatures with fast conformational dynamics—regimes previously inaccessible to mechanistic, single-molecule studies. Here, single-walled carbon nanotube transistors recorded the motions of Taq molecules processing matched or mismatched template–deoxynucleotide triphosphate pairs from 22° to 85°C. By using four enzyme orientations, the whole-enzyme closures of nucleotide incorporations were distinguished from more rapid, 20-μs closures of Taq’s fingers domain testing complementarity and orientation. On average, one transient closure was observed for every nucleotide binding event; even complementary substrate pairs averaged five transient closures between each catalytic incorporation at 72°C. The rate and duration of the transient closures and the catalytic events had almost no temperature dependence, leaving all of Taq’s temperature sensitivity to its rate-determining open state. American Association for the Advancement of Science 2022-03-11 /pmc/articles/PMC8916733/ /pubmed/35275726 http://dx.doi.org/10.1126/sciadv.abl3522 Text en Copyright © 2022 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC). https://creativecommons.org/licenses/by-nc/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial license (https://creativecommons.org/licenses/by-nc/4.0/) , which permits use, distribution, and reproduction in any medium, so long as the resultant use is not for commercial advantage and provided the original work is properly cited.
spellingShingle Physical and Materials Sciences
Turvey, Mackenzie W.
Gabriel, Kristin N.
Lee, Wonbae
Taulbee, Jeffrey J.
Kim, Joshua K.
Chen, Silu
Lau, Calvin J.
Kattan, Rebecca E.
Pham, Jenifer T.
Majumdar, Sudipta
Garcia, Davil
Weiss, Gregory A.
Collins, Philip G.
Single-molecule Taq DNA polymerase dynamics
title Single-molecule Taq DNA polymerase dynamics
title_full Single-molecule Taq DNA polymerase dynamics
title_fullStr Single-molecule Taq DNA polymerase dynamics
title_full_unstemmed Single-molecule Taq DNA polymerase dynamics
title_short Single-molecule Taq DNA polymerase dynamics
title_sort single-molecule taq dna polymerase dynamics
topic Physical and Materials Sciences
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8916733/
https://www.ncbi.nlm.nih.gov/pubmed/35275726
http://dx.doi.org/10.1126/sciadv.abl3522
work_keys_str_mv AT turveymackenziew singlemoleculetaqdnapolymerasedynamics
AT gabrielkristinn singlemoleculetaqdnapolymerasedynamics
AT leewonbae singlemoleculetaqdnapolymerasedynamics
AT taulbeejeffreyj singlemoleculetaqdnapolymerasedynamics
AT kimjoshuak singlemoleculetaqdnapolymerasedynamics
AT chensilu singlemoleculetaqdnapolymerasedynamics
AT laucalvinj singlemoleculetaqdnapolymerasedynamics
AT kattanrebeccae singlemoleculetaqdnapolymerasedynamics
AT phamjenifert singlemoleculetaqdnapolymerasedynamics
AT majumdarsudipta singlemoleculetaqdnapolymerasedynamics
AT garciadavil singlemoleculetaqdnapolymerasedynamics
AT weissgregorya singlemoleculetaqdnapolymerasedynamics
AT collinsphilipg singlemoleculetaqdnapolymerasedynamics