Conformation of a plasmid replication initiator protein affects its proteolysis by ClpXP system

Proteins from the Rep family of DNA replicationinitiators exist mainly as dimers, but only monomers can initiate DNA replicationby interaction with the replicationorigin (ori). In this study, we investigated both the activation (monomerization) and the degradation of the broad-host-range plasmid RK2 replicationinitiation protein TrfA, which we found to be a member of a class of DNA replicationinitiators 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 replicationinitiator activity and stability in E. coli cells are affected by ClpXP system only when the protein sustains dimeric form.