Design of Novel Relaxase Substrates Based on Rolling Circle Replicases for Bioconjugation to DNA Nanostructures

Abstract: 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

Otras publicaciones de la misma revista o congreso con autores/as de la Universidad de Cantabria

 Fuente: PLoS One. 2016 Mar 30;11(3):e0152666

Editorial: Public Library of Science

 Año de publicación: 2016

Nº de páginas: 19

Tipo de publicación: Artículo de Revista

DOI: 10.1371/journal.pone.0152666

ISSN: 1932-6203

Proyecto español: BFU2014-55534-C2-1-P, BFU2014-55534-C2-2-P