The DnaK chaperone uses different mechanisms to promote and inhibit replication of vibrio cholerae chromosome 2

Abstract

Replication of Vibrio cholerae chromosome 2 (Chr2) depends on molecularchaperone Dna K to facilitate binding of the initiator (Rct B) to the replication origin.The binding occurs at two kinds of site, 12-mers and 39-mers, which promoteand inhibit replication, respectively. Here we show that Dna K employs differentmechanisms to enhance the two kinds of binding. We found that mutations in rct Bthat reduce Dna K binding also reduce 12-mer binding and initiation. The initiationdefect is suppressed by second-site mutations that increase 12-mer binding onlymarginally. Instead, they reduce replication inhibitory mechanisms: Rct B dimerizationand 39-mer binding. One suppressing change was in a dimerization domain which isfolded similarly to the initiator of an iteron plasmid—the presumed progenitor of Chr2. In plasmids, Dna K promotes initiation by reducing dimerization. A different mutation was in the 39-mer binding domain of Rct B and inactivated it, indicating analternative suppression mechanism. Paradoxically, although Dna K increases 39-merbinding, the increase was also achieved by inactivating the Dna K binding site of Rct B. This result suggests that the site inhibits the 39-mer binding domain (via autoinhibition)when prevented from binding Dna K. Taken together, our results reveal animportant feature of the transition from plasmid to chromosome: the Chr2 initiatorretains the plasmid-like dimerization domain and its control by chaperones but usesthe chaperones in an unprecedented way to control the inhibitory 39-mer binding. IMPORTANCE The capacity of proteins to undergo remodeling provides opportunitiesto control their function. However, remodeling remains a poorly understood aspect of the structure-function paradigm due to its dynamic nature. Here we have studied remodeling of the initiator of replication of Vibrio cholerae Chr2 by the molecular chaperone, Dna K. We show that Dna K binds to a site on the Chr2 initiator(Rct B) that promotes initiation by reducing the initiator’s propensity to dimerize. Dimerization of the initiator of the putative plasmid progenitor of Chr2 is also reduced by Dna K, which promotes initiation. Paradoxically, the Dna K binding also promotesreplication inhibition by reducing an autoinhibitory activity of Rct B. In the plasmid-to-chromosome transition, it appears that the initiator has acquired an autoinhibitory activity and along with it a new chaperone activity that apparently helps to control replication inhibition independently of replication promotion.