Nearest-neighbor amino acids of specificity-determining residues influence the activity of engineered Cre-type recombinases

Abstract The tyrosine-type site-specific DNA recombinase Cre recombines its target site, loxP, with high activity and specificity without cross-recombining the target sites of highly related recombinases. Understanding how Cre achieves this precision is key to be able to rationally engineer site-spe...

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Autores principales:	Anjali Soni, Martina Augsburg, Frank Buchholz, M. Teresa Pisabarro
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2020
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/5dbe91c778ae4be9a00457b7e08a610c
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spelling	oai:doaj.org-article:5dbe91c778ae4be9a00457b7e08a610c2021-12-02T16:45:46ZNearest-neighbor amino acids of specificity-determining residues influence the activity of engineered Cre-type recombinases10.1038/s41598-020-70867-52045-2322https://doaj.org/article/5dbe91c778ae4be9a00457b7e08a610c2020-08-01T00:00:00Zhttps://doi.org/10.1038/s41598-020-70867-5https://doaj.org/toc/2045-2322Abstract The tyrosine-type site-specific DNA recombinase Cre recombines its target site, loxP, with high activity and specificity without cross-recombining the target sites of highly related recombinases. Understanding how Cre achieves this precision is key to be able to rationally engineer site-specific recombinases (SSRs) for genome editing applications. Previous work has revealed key residues for target site selectivity in the Cre/loxP and the related Dre/rox recombinase systems. However, enzymes in which these residues were changed to the respective counterpart only showed weak activity on the foreign target site. Here, we use molecular modeling and dynamics simulation techniques to comprehensively explore the mechanisms by which these residues determine target recognition in the context of their flanking regions in the protein–DNA interface, and we establish a structure-based rationale for the design of improved recombination activities. Our theoretical models reveal that nearest-neighbors to the specificity-determining residues are important players for enhancing SSR activity on the foreign target site. Based on the established rationale, we design new Cre variants with improved rox recombination activities, which we validate experimentally. Our work provides new insights into the target recognition mechanisms of Cre-like recombinases and represents an important step towards the rational design of SSRs for applied genome engineering.Anjali SoniMartina AugsburgFrank BuchholzM. Teresa PisabarroNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 10, Iss 1, Pp 1-13 (2020)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Anjali Soni Martina Augsburg Frank Buchholz M. Teresa Pisabarro Nearest-neighbor amino acids of specificity-determining residues influence the activity of engineered Cre-type recombinases
description	Abstract The tyrosine-type site-specific DNA recombinase Cre recombines its target site, loxP, with high activity and specificity without cross-recombining the target sites of highly related recombinases. Understanding how Cre achieves this precision is key to be able to rationally engineer site-specific recombinases (SSRs) for genome editing applications. Previous work has revealed key residues for target site selectivity in the Cre/loxP and the related Dre/rox recombinase systems. However, enzymes in which these residues were changed to the respective counterpart only showed weak activity on the foreign target site. Here, we use molecular modeling and dynamics simulation techniques to comprehensively explore the mechanisms by which these residues determine target recognition in the context of their flanking regions in the protein–DNA interface, and we establish a structure-based rationale for the design of improved recombination activities. Our theoretical models reveal that nearest-neighbors to the specificity-determining residues are important players for enhancing SSR activity on the foreign target site. Based on the established rationale, we design new Cre variants with improved rox recombination activities, which we validate experimentally. Our work provides new insights into the target recognition mechanisms of Cre-like recombinases and represents an important step towards the rational design of SSRs for applied genome engineering.
format	article
author	Anjali Soni Martina Augsburg Frank Buchholz M. Teresa Pisabarro
author_facet	Anjali Soni Martina Augsburg Frank Buchholz M. Teresa Pisabarro
author_sort	Anjali Soni
title	Nearest-neighbor amino acids of specificity-determining residues influence the activity of engineered Cre-type recombinases
title_short	Nearest-neighbor amino acids of specificity-determining residues influence the activity of engineered Cre-type recombinases
title_full	Nearest-neighbor amino acids of specificity-determining residues influence the activity of engineered Cre-type recombinases
title_fullStr	Nearest-neighbor amino acids of specificity-determining residues influence the activity of engineered Cre-type recombinases
title_full_unstemmed	Nearest-neighbor amino acids of specificity-determining residues influence the activity of engineered Cre-type recombinases
title_sort	nearest-neighbor amino acids of specificity-determining residues influence the activity of engineered cre-type recombinases
publisher	Nature Portfolio
publishDate	2020
url	https://doaj.org/article/5dbe91c778ae4be9a00457b7e08a610c
work_keys_str_mv	AT anjalisoni nearestneighboraminoacidsofspecificitydeterminingresiduesinfluencetheactivityofengineeredcretyperecombinases AT martinaaugsburg nearestneighboraminoacidsofspecificitydeterminingresiduesinfluencetheactivityofengineeredcretyperecombinases AT frankbuchholz nearestneighboraminoacidsofspecificitydeterminingresiduesinfluencetheactivityofengineeredcretyperecombinases AT mteresapisabarro nearestneighboraminoacidsofspecificitydeterminingresiduesinfluencetheactivityofengineeredcretyperecombinases
_version_	1718383455162597376

Nearest-neighbor amino acids of specificity-determining residues influence the activity of engineered Cre-type recombinases

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