2024-03-28T23:55:53Zhttp://digital.csic.es/dspace-oai/requestoai:digital.csic.es:10261/1644872020-12-13T09:17:44Zcom_10261_3284com_10261_1col_10261_3285
2018-05-07T12:35:21Z
urn:hdl:10261/164487
Design of novel relaxase substrates based on rolling circle replicases for bioconjugation to DNA nanostructures
Sagredo, Sandra
Cruz, Fernando de la
Moncalián, Gabriel
European Commission
Ministerio de Economía y Competitividad (España)
During bacterial conjugation and rolling circle replication, HUH endonucleases, respectively known as relaxases and replicases, form a covalent bond with ssDNA when they cleave their target sequence (nic site). Both protein families show structural similarity but limited amino acid identity. Moreover, the organization of the inverted repeat (IR) and the loop that shape the nic site differs in both proteins. Arguably, replicases cleave their target site more efficiently, while relaxases exert more biochemical control over the process. Here we show that engineering a relaxase target by mimicking the replicase target, results in enhanced formation of protein-DNA covalent complexes. Three widely different relaxases, which belong to MOBF, MOBQ and MOBP families, can properly cleave DNA sequences with permuted target sequences. Collaterally, the secondary structure that the permuted targets acquired within a supercoiled plasmid DNA resulted in poor conjugation frequencies underlying the importance of relaxase accessory proteins in conjugative DNA processing. Our results reveal that relaxase and replicase targets can be interchangeable in vitro. The new Rep substrates provide new bioconjugation tools for the design of sophisticated DNA-protein nanostructures.
2018-05-07T12:35:21Z
2018-05-07T12:35:21Z
2016
2018-05-07T12:35:21Z
artículo
PLoS ONE 11(3): e0152666 (2016)
http://hdl.handle.net/10261/164487
10.1371/journal.pone.0152666
http://dx.doi.org/10.13039/501100003329
http://dx.doi.org/10.13039/501100000780
27027740
eng
Publisher's version
https://doi.org/10.1371/journal.pone.0152666
Sí
info:eu-repo/grantAgreement/MINECO/Plan Estatal de Investigación Científica y Técnica y de Innovación 2013-2016/BFU2014-55534-C2-2-P
info:eu-repo/grantAgreement/EC/FP7/612146
info:eu-repo/grantAgreement/MINECO/Plan Estatal de Investigación Científica y Técnica y de Innovación 2013-2016/BFU2014-55534-C2-1-P
http://creativecommons.org/licenses/by/4.0/
openAccess
Public Library of Science