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Identification of Relaxase-DNA Covalent Complexes and DNA Strand Transfer Reaction Products by Polyacrylamide Gel Electrophoresis

  • María Lucas
  • Gabriel MoncaliánEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2075)

Abstract

Relaxases are essential proteins for plasmid conjugation. They process the DNA to be transferred by means of a covalent intermediate. Thus, the characterization of these covalent complexes is essential to understand the biological role of this reaction and to improve it for biotechnological applications. In this article, we describe the use of the polyacrylamide electrophoresis techniques for the identification of relaxase-DNA covalent complexes, being SDS-PAGE a simple and reliable method for the detection of protein-DNA covalent adducts. Relaxases also perform a strand transfer reaction to recircularize the DNA and finish the DNA transfer process in the recipient cell. Urea-PAGE allows us the analysis of oligonucleotides generated by the strand transfer reaction. These methods could also be used for the analysis of other HUH endonucleases.

Key words

Relaxase HUH endonucleases nic cleavage assay Protein-DNA covalent complex SDS-PAGE 

Notes

Acknowledgments

This work was supported by grants BFU2014-55534-C2-2-P and RYC-2016-20342 from the Spanish Ministry of Economy and Competitiveness. We are grateful to Mapi Garcillán for critical reading of the manuscript.

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  1. 1.Departamento de Biología Molecular, Instituto de Biomedicina y Biotecnología de Cantabria (IBBTEC)Universidad de Cantabria-Consejo Superior de Investigaciones CientíficasSantanderSpain

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