Initiation of replication of plasmid pMV158: mechanisms of DNA strand-transfer reactions mediated by the initiator RepB protein

Journal of Molecular Biology
M MoscosoManuel Espinosa

Abstract

The initiator RepB protein of the rolling circle-replicating plasmid pMV158 has nicking-closing (topoisomerase I-like) activities on supercoiled DNA. RepB is also able to perform a strand-transfer reaction on a single-stranded DNA substrate that contains its target. Several attempts at capturing covalent protein-DNA intermediates were made to identify the mechanism of RepB-mediated activity. Whereas RepB did not generate stable complexes with its target DNA, employment of single-stranded oligonucleotides containing a chiral phosphorothioate in the target DNA allowed us to follow the process of RepB-mediated strand-transfer reaction. This reaction occurred through a number of even steps because the chirality of the phosphorothioate at the reaction site was retained after RepB-mediated strand transfer. This finding suggests the existence of a covalent intermediate during the strand-transfer reaction between the protein and its target DNA. By site-directed mutagenesis at the codon for Tyr99 of RepB, and purification and assay of activity of the mutant protein variants, we showed that the Tyr99 residue is involved in the nucleophilic attack of RepB to its cognate DNA.

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Citations

Aug 2, 2013·Proceedings of the National Academy of Sciences of the United States of America·María Victoria FranciaGabriel Moncalián
Nov 26, 2010·Molecular Biology and Evolution·Jörn PetersenSilke Pradella
Jun 28, 2000·Nucleic Acids Research·S MarsinP Forterre
Jul 25, 2012·Genome Research·Tim van Opijnen, Andrew Camilli
Oct 7, 2016·Frontiers in Molecular Biosciences·José A Ruiz-MasóGloria Del Solar
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Mar 5, 2011·Archives of Microbiology·Jörn Petersen
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Jan 17, 2004·Biochimica Et Biophysica Acta·José A Ruiz-MasóGloria del Solar
Dec 9, 2021·Microbiology and Molecular Biology Reviews : MMBR·M Pilar Garcillán-BarciaManuel Espinosa

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