Structural features of the initiator of replication protein RepB encoded by the promiscuous plasmid pMV158.

Abstract The promiscuous rolling circle (RC)-replicating plasmid pMV158 encodes the 210-amino-acid initiator of replication protein, RepB. The protein relaxes supercoiled cognate DNA in a topoisomeraseI-like manner. A new vector and procedure for overproduction, scaling-up, and purification of the protein has been developed. RepB purified as a hexamer in solution, as shown by analytical ultracentrifugation assays. Circular dichroism (CD) of RepB indicated that the protein has an estimated content of around 33% α-helices and 20% β-strands. Characterisation of temperature-induced transitions of the protein showed an irreversible change in the spectra when the temperature was raised above 35 °C, indicating that the protein undergoes a conformational change that could account for the relatively high optimal temperature of the RepB-mediated cleavage.