title: 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.
authors.affiliation: Departamento de Biología Molecular e Instituto de Biomedicina y Biotecnología de Cantabria, Universidad de Cantabria-Consejo Superior de Investigaciones Científicas-SODERCAN, C/ Albert Einstein 22, 39011, Santander, Spain.
authors.forename: Sandra
authors.initials: S
authors.lastname: Sagredo
authors.affiliation: Departamento de Biología Molecular e Instituto de Biomedicina y Biotecnología de Cantabria, Universidad de Cantabria-Consejo Superior de Investigaciones Científicas-SODERCAN, C/ Albert Einstein 22, 39011, Santander, Spain.
authors.forename: Fernando
authors.initials: F
authors.lastname: de la Cruz
authors.affiliation: Departamento de Biología Molecular e Instituto de Biomedicina y Biotecnología de Cantabria, Universidad de Cantabria-Consejo Superior de Investigaciones Científicas-SODERCAN, C/ Albert Einstein 22, 39011, Santander, Spain.
authors.forename: Gabriel
authors.initials: G
authors.lastname: Moncalián
mesh_terms: D004265:DNA Helicases / Q000737:chemistry* / Q000378:metabolism; D004269:DNA, Bacterial / Q000737:chemistry* / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000737:chemistry* / Q000378:metabolism; D004278:DNA, Superhelical / Q000737:chemistry / Q000378:metabolism; D004706:Endodeoxyribonucleases / Q000737:chemistry* / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry* / Q000378:metabolism; D055029:Inverted Repeat Sequences; D049329:Nanostructures / Q000737:chemistry*; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D015534:Trans-Activators / Q000737:chemistry* / Q000378:metabolism
chemical_list: D004269:DNA, Bacterial; D004277:DNA, Single-Stranded; D004278:DNA, Superhelical; D015534:Trans-Activators; C045264:replication initiator protein; D004706:Endodeoxyribonucleases; D004265:DNA Helicases