In Vivo and In Vitro Studies of a Copy Number Mutation of the RepA Replication Protein of Plasmid pSC101

Article Article scientifique Article In Vivo and In Vitro Studies of a Copy Number Mutation of the RepA Replication Protein of Plasmid pSC101 eng 1993 author Xia Gueixian 22413 author Manen-Commandeur Danielle 22369 author Yu Yi-Yi 73629 author Caro Lucien 25084 4165-4175 10.1128/JB.175.13.4165-4175.1993 0021-9193 https://JB.asm.org/content/175/13/4165 The RepA replication protein of plasmid pSC101 binds as a monomer to three repeated sequences (RS1, RS2, and RS3) in the replication origin of the plasmid to initiate duplication and binds as a dimer to two inversely repeated sequences (IR1 and IR2) in its promoter region (D. Manen, 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. Biol. 181:383-393, 1985). A mutation in the protein RepA(cop) that affects a single amino acid increases the plasmid copy number fourfold. In vivo experiments show that, when provided in trans under a foreign promoter, the RepA(cop) protein increases the replication of a plasmid containing the origin of replication without repA, whereas it decreases the repression of its own promoter. In vitro experiments show that the purified RepA(cop) protein binds more efficiently to the repeated sequences 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: the wild-type protein binds efficiently to that sequence, whereas the mutated protein binds less efficiently and less specifically. Footprint experiments confirmed these results and, in addition, showed a difference in the pattern of protection of the inversely repeated sequences by the mutant protein. Equilibrium binding experiments showed that the formation of protein-probe complexes at increasing concentrations of protein had a sigmoidal shape for binding to RS sequences and a hyperbolic shape for binding to IR sequences. The results, together with earlier work (G.-X. Xia, D. Manen, T. Goebel, P. Linder, G. Churchward, and 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 plasmid replication and that its binding to IR sequences plays a role in the autoregulation of RepA expression. They also demonstrate that the two separate functions of the protein are effected by two different forms of binding to the target sites. Dewey 570 Swiss National Science Foundation 31-25698.88 CH Autre Public Health Service grant GM36437 from the National Institutes of Health 43 Département de biologie moléculaire 161 false Journal of Bacteriology 175 13 7a2f17ba-3774-4348-abed-017275eb2665 Article (Published version) Restricted JB-1993-Xia-4165.full.pdf 2715230 application/pdf restrictedAccess Faculté des sciences / Section de biologie / Département de biologie moléculaire

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In Vivo and In Vitro Studies of a Copy Number Mutation of the RepA Replication Protein of Plasmid pSC101	Journal of Bacteriology			1993	286	0

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Yu, Yi-Yi