Cargando…
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...
Autores principales: | , , , , , , |
---|---|
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 |
_version_ | 1782238601249030144 |
---|---|
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. |
format | Online Article Text |
id | pubmed-3401438 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2012 |
publisher | Oxford University Press |
record_format | MEDLINE/PubMed |
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 |
work_keys_str_mv | AT manosasmaria mechanismofstranddisplacementsynthesisbydnareplicativepolymerases AT spieringmichellem mechanismofstranddisplacementsynthesisbydnareplicativepolymerases AT dingfangyuan mechanismofstranddisplacementsynthesisbydnareplicativepolymerases AT bensimondavid mechanismofstranddisplacementsynthesisbydnareplicativepolymerases AT allemandjeanfrancois mechanismofstranddisplacementsynthesisbydnareplicativepolymerases AT benkovicstephenj mechanismofstranddisplacementsynthesisbydnareplicativepolymerases AT croquettevincent mechanismofstranddisplacementsynthesisbydnareplicativepolymerases |