IN-VIVO AND IN-VITRO STUDIES OF A COPY NUMBER MUTATION OF THE REPA REPLICATION PROTEIN OF PLASMID-PSC101

The RepA replication protein of plasmid pSC101 binds as a monomer to t hree repeated sequences (RS1, RS2, and RS3) in the replication origin of the plasmid to initiate duplication and binds as a dimer to two inv ersely repeated sequences (IRI and IR2) in its promoter region (D. Man en, L. C. Upegui-Gonzalez, and L. Caro, Proc. Natl. Acad. Sci. USA 89: 8923-8927, 1992). The binding to IR2 autoregulates repA transcription (P. Linder, G. Churchward, G. X. Xia, Y. Y. Yu, and L. Caro, J. Mol. B iol. 181:383-393, 1985). A mutation in the protein RepA(cop) that affe cts a single amino acid increases the plasmid copy number fourfold. In vivo experiments show that, when provided in trans under a foreign pr omoter, the RepA(cop) protein increases the replication of a plasmid c ontaining the origin of replication without repA, whereas it decreases the repression of its own promoter. In vitro experiments show that th e purified RepA(cop) protein binds more efficiently to the repeated se quences within the origin than does RepA and that its binding to these sequences is more specific than that of RepA. Binding to an inversely repeated sequence within the repA promoter gives opposite results: th e wild-type protein binds efficiently to that sequence, whereas the mu tated protein binds less efficiently and less specifically. Footprint experiments confirmed these results and, in addition, showed a differe nce in the pattern of protection of the inversely repeated sequences b y the mutant protein. Equilibrium binding experiments showed that the formation of protein-probe complexes at increasing concentrations of p rotein had a sigmoidal shape for binding to RS sequences and a hyperbo lic shape for binding to IR sequences. The results, together with earl ier work (G.-X. Xia, D. Manen, T. Goebel, P. Linder, G. Churchward, an d L. Caro, Mol. Microbiol. 5:631-640, 1991), confirm that the binding of RepA to RS sequences plays a crucial role in the regulation of plas mid replication and that its binding to IR sequences plays a role in t he autoregulation of RepA expression. They also demonstrate that the t wo separate functions of the protein are effected by two different for ms of binding to the target sites.