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Purification of the beta product encoded by the Streptococcus pyogenes plasmid pSM19035. A putative DNA recombinase
1Centro de Biología Molecular Severo Ochoa, UAM-CSIC, Madrid, Spain.
Abstract:
Genetic evidence suggests that the gene beta product of Streptococcus pyogenes plasmid pSM19035 is required for converting plasmid multimers into monomers. The beta protein was purified from cells overexpressing the cloned gene. N-terminal protein sequence analysis demonstrated that the purified protein had the predicted sequence, except that the N-terminal initiator methionine was not present. Native beta protein consists of a dimer of two identical subunits with a molecular mass of 23.8 kDa (25 kDa in SDS-PAGE). The beta protein (isoelectric point of 9.7) binds specifically to a DNA fragment (312 bp in length) which contains the promoter region of the orf alpha-gene beta operon and two regions (sites I and II) that show dyad axes of symmetry. It is proposed that protein beta binds to sites I and II to mediate resolution of plasmid oligomers.
Insights
Streptococcus pyogenes beta protein resolves plasmid multimers into monomers. This essential protein binds specific DNA sites to mediate this crucial plasmid replication process.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Plasmid replication involves complex mechanisms, including the resolution of multimeric forms into monomers.
- Streptococcus pyogenes plasmid pSM19035 harbors genes essential for its maintenance and replication.
- The beta protein is implicated in the conversion of plasmid multimers to monomers.
Purpose of the Study:
- To characterize the beta protein from Streptococcus pyogenes plasmid pSM19035.
- To investigate the DNA binding properties and function of the beta protein in plasmid replication.
Main Methods:
- Overexpression and purification of the beta protein.
- N-terminal protein sequencing and SDS-PAGE for molecular mass determination.
- DNA binding assays using a specific DNA fragment containing promoter and dyad symmetry regions.
Main Results:
- The beta protein was purified, confirming its predicted sequence (minus initiator methionine).
- Native beta protein exists as a dimer (23.8 kDa), with a 25 kDa size in SDS-PAGE.
- The beta protein specifically binds to a 312 bp DNA fragment containing the orf alpha-gene beta operon promoter and two symmetrical sites (I and II).
Conclusions:
- The beta protein is a DNA-binding protein crucial for resolving plasmid multimers into monomers in Streptococcus pyogenes.
- Binding to specific DNA sites (I and II) is proposed as the mechanism for mediating plasmid oligomer resolution.