The DnaK-DnaJ-GrpE chaperone system activates inert wild type π initiator protein of R6K into a form active in replication initiation

Abstract

The plasmid R6K is an interesting model system for investigating initiation of DNA replication, not only near the primary binding sites of the initiator protein π but also at a distance, caused by π-mediated DNA looping. An important milestone in the mechanistic analysis of this replicon was the development of a reconstituted replication system consisting of 22 different highly purified proteins (Abhyankar, M. A., Zzaman, S., and Bastia, D. (2003) J. Biol. Chem. 278, 45476-45484). Although the in vitro reconstituted system promotes ori γ-specific initiation of replication by a mutant form of the initiator called π*, the wild type (WT) π is functionally inert in this system. Here we show that the chaperone DnaK along with its co-chaperone DnaJ and the nucleotide exchange factor GrpE were needed to activate WT π and caused it to initiate replication in vitro at the correct origin. We show further that the reaction was relatively chaperone-specific and that other chaperones, such as ClpB and ClpX, were incapable of activating WT π. The molecular mechanism of activation appeared to be a chaperone-catalyzed facilitation of dimeric inert WT π into iteron-bound monomers. Protein-protein interaction analysis by enzyme-linked immunosorbent assay revealed that, in the absence of ATP, DnaJ directly interacted with π but its binary interactions with DnaK and GrpE and with ClpB and ClpX were at background levels, suggesting that π recruited by protein-protein interaction with DnaJ and then fed into the DnaK chaperone machine to promote initiator activation.