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Characterization of the tyrosine recombinase MrpA encoded by the Streptomyces coelicolor A3(2) plasmid SCP2*
Lydia Warth1, Iris Haug, Josef Altenbuchner
1Institut für Industrielle Genetik, Universität Stuttgart, Allmandring 31, 70569 Stuttgart, Germany.
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.
Insights
MrpA is identified as a novel site-specific tyrosine recombinase. This protein, along with its binding site mrpS, mediates DNA integration, resolution, and inversion without needing accessory factors.
Area of Science:
- Molecular biology
- Genetics
- Biochemistry
Background:
- MrpA is the multimer resolution protein of the Streptomyces coelicolor A3(2) plasmid SCP2*.
- Previous studies indicated MrpA enhances the stability of SCP2*-derived plasmids in Streptomyces lividans.
Purpose of the Study:
- To functionally characterize MrpA and elucidate its mechanism of action.
- To identify the DNA binding site of MrpA and determine its recombinase activity.
Main Methods:
- Sequence alignment to identify conserved residues.
- Overexpression and purification of MrpA with Strep-tag.
- DNase I footprinting and gel mobility shift assays to identify the MrpA binding site (mrpS).
- In vivo and in vitro assays to assess MrpA activity.
- Site-directed mutagenesis to investigate the role of conserved tyrosine (Y354).
Main Results:
- Sequence alignment revealed MrpA shares conserved residues with tyrosine recombinases.
- The 36-bp binding site, mrpS, was identified, featuring a configuration typical for tyrosine recombinases.
- MrpA demonstrated integration, resolution, and inversion activities depending on mrpS site arrangement, without requiring accessory factors.
- Mutating the conserved tyrosine (Y354F) abolished recombination activity while maintaining DNA binding.
Conclusions:
- MrpA is defined as a new site-specific tyrosine recombinase that functions with its binding site, mrpS.
- The conserved tyrosine residue Y354 is crucial for the DNA cleavage activity during recombination.
- MrpA represents a novel tool for manipulating DNA structures in biotechnological applications.
