Conformation of a plasmid replication initiator protein affects its proteolysis by ClpXP system |
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| Authors: | Marcin Pierechod Agnieszka Nowak Anna Saari Elzbieta Purta Janusz M Bujnicki Igor Konieczny |
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| Institution: | 1. Department of Molecular and Cellular Biology, Faculty of Biotechnology, University of Gdansk, 80‐822 Gdansk, Poland;2. Laboratory of Bioinformatics and Protein Engineering, International Institute of Molecular and Cell Biology, 02‐109 Warsaw, Poland;3. PhD school, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 02‐106 Warsaw, Poland;4. Laboratory of Bioinformatics, Institute of Molecular Biology and Biotechnology, Faculty of Biology, Adam Mickiewicz University, 61‐614 Poznan, Poland |
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| Abstract: | Proteins from the Rep family of DNA replication initiators exist mainly as dimers, but only monomers can initiate DNA replication by interaction with the replication origin (ori). In this study, we investigated both the activation (monomerization) and the degradation of the broad‐host‐range plasmid RK2 replication initiation protein TrfA, which we found to be a member of a class of DNA replication initiators containing winged helix (WH) domains. Our in vivo and in vitro experiments demonstrated that the ClpX‐dependent activation of TrfA leading to replicationally active protein monomers and mutations affecting TrfA dimer formation, result in the inhibition of TrfA protein degradation by the ClpXP proteolytic system. These data revealed that the TrfA monomers and dimers are degraded at substantially different rates. Our data also show that the plasmid replication initiator activity and stability in E. coli cells are affected by ClpXP system only when the protein sustains dimeric form. |
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| Keywords: | structure/function studies DNA‐binding domains circular dichroism computational analysis of protein structure protein structure prediction Rep proteins protein stability proteolysis |
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