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Mechanism of strand displacement synthesis by DNA replicative polymerases

Replicative holoenzymes exhibit rapid and processive primer extension DNA synthesis, but inefficient strand displacement DNA synthesis. We investigated the bacteriophage T4 and T7 holoenzymes primer extension activity and strand displacement activity on a DNA hairpin substrate manipulated by a magne...

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Autores principales: Manosas, Maria, Spiering, Michelle M., Ding, Fangyuan, Bensimon, David, Allemand, Jean-François, Benkovic, Stephen J., Croquette, Vincent
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2012
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3401438/
https://www.ncbi.nlm.nih.gov/pubmed/22434889
http://dx.doi.org/10.1093/nar/gks253
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author Manosas, Maria
Spiering, Michelle M.
Ding, Fangyuan
Bensimon, David
Allemand, Jean-François
Benkovic, Stephen J.
Croquette, Vincent
author_facet Manosas, Maria
Spiering, Michelle M.
Ding, Fangyuan
Bensimon, David
Allemand, Jean-François
Benkovic, Stephen J.
Croquette, Vincent
author_sort Manosas, Maria
collection PubMed
description Replicative holoenzymes exhibit rapid and processive primer extension DNA synthesis, but inefficient strand displacement DNA synthesis. We investigated the bacteriophage T4 and T7 holoenzymes primer extension activity and strand displacement activity on a DNA hairpin substrate manipulated by a magnetic trap. Holoenzyme primer extension activity is moderately hindered by the applied force. In contrast, the strand displacement activity is strongly stimulated by the applied force; DNA polymerization is favoured at high force, while a processive exonuclease activity is triggered at low force. We propose that the DNA fork upstream of the holoenzyme generates a regression pressure which inhibits the polymerization-driven forward motion of the holoenzyme. The inhibition is generated by the distortion of the template strand within the polymerization active site thereby shifting the equilibrium to a DNA-protein exonuclease conformation. We conclude that stalling of the holoenzyme induced by the fork regression pressure is the basis for the inefficient strand displacement synthesis characteristic of replicative polymerases. The resulting processive exonuclease activity may be relevant in replisome disassembly to reset a stalled replication fork to a symmetrical situation. Our findings offer interesting applications for single-molecule DNA sequencing.
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spelling pubmed-34014382012-07-23 Mechanism of strand displacement synthesis by DNA replicative polymerases Manosas, Maria Spiering, Michelle M. Ding, Fangyuan Bensimon, David Allemand, Jean-François Benkovic, Stephen J. Croquette, Vincent Nucleic Acids Res Nucleic Acid Enzymes Replicative holoenzymes exhibit rapid and processive primer extension DNA synthesis, but inefficient strand displacement DNA synthesis. We investigated the bacteriophage T4 and T7 holoenzymes primer extension activity and strand displacement activity on a DNA hairpin substrate manipulated by a magnetic trap. Holoenzyme primer extension activity is moderately hindered by the applied force. In contrast, the strand displacement activity is strongly stimulated by the applied force; DNA polymerization is favoured at high force, while a processive exonuclease activity is triggered at low force. We propose that the DNA fork upstream of the holoenzyme generates a regression pressure which inhibits the polymerization-driven forward motion of the holoenzyme. The inhibition is generated by the distortion of the template strand within the polymerization active site thereby shifting the equilibrium to a DNA-protein exonuclease conformation. We conclude that stalling of the holoenzyme induced by the fork regression pressure is the basis for the inefficient strand displacement synthesis characteristic of replicative polymerases. The resulting processive exonuclease activity may be relevant in replisome disassembly to reset a stalled replication fork to a symmetrical situation. Our findings offer interesting applications for single-molecule DNA sequencing. Oxford University Press 2012-07 2012-03-20 /pmc/articles/PMC3401438/ /pubmed/22434889 http://dx.doi.org/10.1093/nar/gks253 Text en © The Author(s) 2012. Published by Oxford University Press. http://creativecommons.org/licenses/by-nc/3.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Nucleic Acid Enzymes
Manosas, Maria
Spiering, Michelle M.
Ding, Fangyuan
Bensimon, David
Allemand, Jean-François
Benkovic, Stephen J.
Croquette, Vincent
Mechanism of strand displacement synthesis by DNA replicative polymerases
title Mechanism of strand displacement synthesis by DNA replicative polymerases
title_full Mechanism of strand displacement synthesis by DNA replicative polymerases
title_fullStr Mechanism of strand displacement synthesis by DNA replicative polymerases
title_full_unstemmed Mechanism of strand displacement synthesis by DNA replicative polymerases
title_short Mechanism of strand displacement synthesis by DNA replicative polymerases
title_sort mechanism of strand displacement synthesis by dna replicative polymerases
topic Nucleic Acid Enzymes
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3401438/
https://www.ncbi.nlm.nih.gov/pubmed/22434889
http://dx.doi.org/10.1093/nar/gks253
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