Cargando…
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...
Autores principales: | , , , , , |
---|---|
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 |
_version_ | 1783528046036254720 |
---|---|
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. |
format | Online Article Text |
id | pubmed-7192630 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2020 |
publisher | Oxford University Press |
record_format | MEDLINE/PubMed |
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 |
work_keys_str_mv | AT shouramassaj loopclosurekineticsrevealastablerighthandeddnaintermediateincrerecombination AT giovanstefanm loopclosurekineticsrevealastablerighthandeddnaintermediateincrerecombination AT vetcheralexandrea loopclosurekineticsrevealastablerighthandeddnaintermediateincrerecombination AT ziraldoriccardo loopclosurekineticsrevealastablerighthandeddnaintermediateincrerecombination AT hankeandreas loopclosurekineticsrevealastablerighthandeddnaintermediateincrerecombination AT levenestephend loopclosurekineticsrevealastablerighthandeddnaintermediateincrerecombination |