title: Characterization of the tyrosine recombinase MrpA encoded by the Streptomyces coelicolor A3(2) plasmid SCP2*.
abstract: MrpA is the multimer resolution protein of the Streptomyces coelicolor A3(2) plasmid SCP2*. Previously, MrpA was found to significantly increase the stability of SCP2*-derived plasmids in Streptomyces lividans. The present report gives a functional characterization of MrpA. A sequence alignment revealed that MrpA shares highly conserved residues with members of the tyrosine recombinase family. After overexpression and Strep-tag purification, a DNase I footprint analysis and a gel mobility shift assay allowed for the identification of the 36-bp MrpA binding site mrpS. The mrpS site shows the configuration typical for tyrosine recombinases and contains two MrpA binding sites. The activity of MrpA was explored in vivo in E. coli cells and in vitro using purified MrpA. Depending on the position and orientation of the mrpS sites, three activities were detected: integration, resolution, and inversion. No accessory sites or proteins were required. Substitution of the conserved tyrosine (Y354F) by site-directed mutagenesis resulted in a complete loss of recombination activity but it still allowed the binding of MrpA to mrpS. The results define MrpA as a new site-specific tyrosine recombinase that acts with mrpS. In addition, we suggest that Y354 provides the nucleophile for DNA cleavage during recombination.
authors.affiliation: Institut für Industrielle Genetik, Universität Stuttgart, Allmandring 31, 70569 Stuttgart, Germany.
authors.forename: Lydia
authors.initials: L
authors.lastname: Warth
authors.forename: Iris
authors.initials: I
authors.lastname: Haug
authors.forename: Josef
authors.initials: J
authors.lastname: Altenbuchner
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D018983:DNA Footprinting; D004254:DNA Nucleotidyltransferases / Q000235:genetics / Q000378:metabolism*; D004269:DNA, Bacterial / Q000235:genetics; D024202:Electrophoretic Mobility Shift Assay; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids / Q000235:genetics; D016415:Sequence Alignment; D048372:Streptomyces coelicolor / Q000201:enzymology* / Q000235:genetics; D014443:Tyrosine / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D014443:Tyrosine; D004254:DNA Nucleotidyltransferases; C077570:Site-specific recombinase