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A novel site-specific recombinase encoded by the Streptococcus pyogenes plasmid pSM19035
1Centro de Biología Molecular Severo Ochoa, C.S.I.C., Universidad Autónoma de Madrid, Spain.
Journal of Molecular Biology
|April 29, 1994
Summary
Streptococcus pyogenes beta protein resolves plasmid DNA multimers and inverts DNA segments, but requires host factors for recombination. It binds specific DNA sites, forming dimers that may interact, with host factors potentially compensating for a missing binding site.
Area of Science:
- Molecular Biology
- Genetics
- Microbiology
Background:
- The beta protein from Streptococcus pyogenes plasmid pSM19035 is implicated in plasmid DNA resolution and inversion.
- Previous genetic evidence suggested a role for beta protein in these DNA recombination processes.
Purpose of the Study:
- To investigate the in vitro DNA recombination activity of purified beta protein.
- To characterize the DNA binding properties and requirements of the beta protein.
Main Methods:
- In vitro DNA recombination assays using supercoiled templates.
- DNase I footprinting to map protein binding sites.
- Gel retardation assays to study protein-DNA interactions.
Main Results:
- Purified beta protein requires host factor(s) to catalyze DNA recombination.
- Beta protein mediates intramolecular recombination, resulting in DNA resolution (direct repeat orientation) or inversion (inverse repeat orientation).
- Beta protein binds to a specific 85 bp DNA region containing two discrete sites (I and II), forming a complex likely composed of two dimers.
Conclusions:
- Host factors are essential for beta protein-mediated DNA recombination.
- The beta protein binding site differs from related DNA resolvases, possessing two sites instead of three.
- Host factors may act as accessory effectors, compensating for the reduced number of binding sites on the DNA substrate.