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Loop-closure kinetics reveal a stable, right-handed DNA intermediate in Cre recombination

In Cre site-specific recombination, the synaptic intermediate is a recombinase homotetramer containing a pair of loxP DNA target sites. The enzyme system's strand-exchange mechanism proceeds via a Holliday-junction (HJ) intermediate; however, the geometry of DNA segments in the synapse has rema...

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Autores principales: Shoura, Massa J, Giovan, Stefan M, Vetcher, Alexandre A, Ziraldo, Riccardo, Hanke, Andreas, Levene, Stephen D
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7192630/
https://www.ncbi.nlm.nih.gov/pubmed/32182357
http://dx.doi.org/10.1093/nar/gkaa153
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author Shoura, Massa J
Giovan, Stefan M
Vetcher, Alexandre A
Ziraldo, Riccardo
Hanke, Andreas
Levene, Stephen D
author_facet Shoura, Massa J
Giovan, Stefan M
Vetcher, Alexandre A
Ziraldo, Riccardo
Hanke, Andreas
Levene, Stephen D
author_sort Shoura, Massa J
collection PubMed
description In Cre site-specific recombination, the synaptic intermediate is a recombinase homotetramer containing a pair of loxP DNA target sites. The enzyme system's strand-exchange mechanism proceeds via a Holliday-junction (HJ) intermediate; however, the geometry of DNA segments in the synapse has remained highly controversial. In particular, all crystallographic structures are consistent with an achiral, planar Holliday-junction (HJ) structure, whereas topological assays based on Cre-mediated knotting of plasmid DNAs are consistent with a right-handed chiral junction. We use the kinetics of loop closure involving closely spaced (131–151 bp) loxP sites to investigate the in-aqueo ensemble of conformations for the longest-lived looped DNA intermediate. Fitting the experimental site-spacing dependence of the loop-closure probability, J, to a statistical-mechanical theory of DNA looping provides evidence for substantial out-of-plane HJ distortion, which unequivocally stands in contrast to the square-planar intermediate geometry from Cre-loxP crystal structures and those of other int-superfamily recombinases. J measurements for an HJ-isomerization-deficient Cre mutant suggest that the apparent geometry of the wild-type complex is consistent with temporal averaging of right-handed and achiral structures. Our approach connects the static pictures provided by crystal structures and the natural dynamics of macromolecules in solution, thus advancing a more comprehensive dynamic analysis of large nucleoprotein structures and their mechanisms.
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spelling pubmed-71926302020-05-06 Loop-closure kinetics reveal a stable, right-handed DNA intermediate in Cre recombination Shoura, Massa J Giovan, Stefan M Vetcher, Alexandre A Ziraldo, Riccardo Hanke, Andreas Levene, Stephen D Nucleic Acids Res Molecular Biology In Cre site-specific recombination, the synaptic intermediate is a recombinase homotetramer containing a pair of loxP DNA target sites. The enzyme system's strand-exchange mechanism proceeds via a Holliday-junction (HJ) intermediate; however, the geometry of DNA segments in the synapse has remained highly controversial. In particular, all crystallographic structures are consistent with an achiral, planar Holliday-junction (HJ) structure, whereas topological assays based on Cre-mediated knotting of plasmid DNAs are consistent with a right-handed chiral junction. We use the kinetics of loop closure involving closely spaced (131–151 bp) loxP sites to investigate the in-aqueo ensemble of conformations for the longest-lived looped DNA intermediate. Fitting the experimental site-spacing dependence of the loop-closure probability, J, to a statistical-mechanical theory of DNA looping provides evidence for substantial out-of-plane HJ distortion, which unequivocally stands in contrast to the square-planar intermediate geometry from Cre-loxP crystal structures and those of other int-superfamily recombinases. J measurements for an HJ-isomerization-deficient Cre mutant suggest that the apparent geometry of the wild-type complex is consistent with temporal averaging of right-handed and achiral structures. Our approach connects the static pictures provided by crystal structures and the natural dynamics of macromolecules in solution, thus advancing a more comprehensive dynamic analysis of large nucleoprotein structures and their mechanisms. Oxford University Press 2020-05-07 2020-03-17 /pmc/articles/PMC7192630/ /pubmed/32182357 http://dx.doi.org/10.1093/nar/gkaa153 Text en © The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research. http://creativecommons.org/licenses/by-nc/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com
spellingShingle Molecular Biology
Shoura, Massa J
Giovan, Stefan M
Vetcher, Alexandre A
Ziraldo, Riccardo
Hanke, Andreas
Levene, Stephen D
Loop-closure kinetics reveal a stable, right-handed DNA intermediate in Cre recombination
title Loop-closure kinetics reveal a stable, right-handed DNA intermediate in Cre recombination
title_full Loop-closure kinetics reveal a stable, right-handed DNA intermediate in Cre recombination
title_fullStr Loop-closure kinetics reveal a stable, right-handed DNA intermediate in Cre recombination
title_full_unstemmed Loop-closure kinetics reveal a stable, right-handed DNA intermediate in Cre recombination
title_short Loop-closure kinetics reveal a stable, right-handed DNA intermediate in Cre recombination
title_sort loop-closure kinetics reveal a stable, right-handed dna intermediate in cre recombination
topic Molecular Biology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7192630/
https://www.ncbi.nlm.nih.gov/pubmed/32182357
http://dx.doi.org/10.1093/nar/gkaa153
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