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.
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References
Chandler M, de la Cruz F, Dyda F, Hickman AB, Moncalian G, Ton-Hoang B (2013) Breaking and joining single-stranded DNA: the HUH endonuclease superfamily. Nat Rev Microbiol 11:525–538. https://doi.org/10.1038/nrmicro3067
Zechner EL, Moncalián G, de la Cruz F (2017) Relaxases and plasmid transfer in gram-negative Bacteria. In: Backert S, Grohmann E (eds) Type IV secretion in gram-negative and gram-positive bacteria. Springer International Publishing, Cham, pp 93–113
Byrd DR, Matson SW (1997) Nicking by transesterification: the reaction catalysed by a relaxase. Mol Microbiol 25:1011–1022. https://doi.org/10.1046/j.1365-2958.1997.5241885.x
Goessweiner-Mohr N, Arends K, Keller W, Grohmann E (2014) Conjugation in gram-positive Bacteria. Microbiol Spectr 2:PLAS-0004-2013. https://doi.org/10.1128/microbiolspec.PLAS-0004-2013
Garcillán-Barcia MP, Francia MV, de la Cruz F (2009) The diversity of conjugative relaxases and its application in plasmid classification. FEMS Microbiol Rev 33:657–687. https://doi.org/10.1111/j.1574-6976.2009.00168.x
Guasch A, Lucas M, Moncalián G, Cabezas M, Pérez-Luque R, Gomis-Rüth FX, de la Cruz F, Coll M (2003) Recognition and processing of the origin of transfer DNA by conjugative relaxase TrwC. Nat Struct Biol 10:1002–1010. https://doi.org/10.1038/nsmb0104-102a
Datta S, Larkin C, Schildbach JF (2003) Structural insights into single-stranded DNA binding and cleavage by F factor TraI. Structure 11:1369–1379. https://doi.org/10.1016/j.str.2003.10.001
Nash RP, Habibi S, Cheng Y, Lujan SA, Redinbo MR (2010) The mechanism and control of DNA transfer by the conjugative relaxase of resistance plasmid pCU1. Nucleic Acids Res 38:5929–5943. https://doi.org/10.1093/nar/gkq303
Monzingo AF, Ozburn A, Xia S, Meyer RJ, Robertus JD (2007) The structure of the minimal relaxase domain of MobA at 2.1 a resolution. J Mol Biol 366:165–178. https://doi.org/10.1016/j.jmb.2006.11.031
Edwards JS, Betts L, Frazier ML, Pollet RM, Kwong SM, Walton WG, Ballentine WK, Huang JJ, Habibi S, Del Campo M, Meier JL, Dervan PB, Firth N, Redinbo MR (2013) Molecular basis of antibiotic multiresistance transfer in Staphylococcus aureus. Proc Natl Acad Sci U S A 110:2804–2809. https://doi.org/10.1073/pnas.1219701110
Miroux B, Walker JE (1996) Over-production of proteins in Escherichia coli: mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels. J Mol Biol 260:289–298. https://doi.org/10.1006/jmbi.1996.0399
Llosa M, Zunzunegui S, de la Cruz F (2003) Conjugative coupling proteins interact with cognate and heterologous VirB10-like proteins while exhibiting specificity for cognate relaxosomes. Proc Natl Acad Sci U S A 100:10465–10470. https://doi.org/10.1073/pnas.1830264100
Gonzalez-Perez B, Lucas M, Cooke LA, Vyle JS, de la Cruz F, Moncalián G (2007) Analysis of DNA processing reactions in bacterial conjugation by using suicide oligonucleotides. EMBO J 26:3847–3857. https://doi.org/10.1038/sj.emboj.7601806
Sagredo S, de la Cruz F, Moncalián G (2016) Design of novel relaxase substrates based on rolling circle replicases for bioconjugation to DNA nanostructures. PLoS One 11:e0152666. https://doi.org/10.1371/journal.pone.0152666
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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Lucas, M., Moncalián, G. (2020). Identification of Relaxase-DNA Covalent Complexes and DNA Strand Transfer Reaction Products by Polyacrylamide Gel Electrophoresis. In: de la Cruz, F. (eds) Horizontal Gene Transfer. Methods in Molecular Biology, vol 2075. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9877-7_11
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DOI: https://doi.org/10.1007/978-1-4939-9877-7_11
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