Abstract
ABSTRACTReplication of Vibrio cholerae chromosome 2 (Chr2) depends on molecular chaperone DnaK to facilitate binding of the initiator (RctB) to the replication origin. The binding occurs at two kinds of site, 12-mers and 39-mers, which promote and inhibit replication, respectively. Here we show that DnaK employs different mechanisms to enhance the two kinds of binding. We found that mutations in rctB that reduce DnaK binding also reduce 12-mer binding and initiation. The initiation defect is suppressed by second-site mutations that increase 12-mer binding only marginally. Instead, they reduce replication inhibitory mechanisms: RctB dimerization and 39-mer binding. One suppressing change was in a dimerization domain which is folded similarly to the initiator of an iteron plasmid—the presumed progenitor of Chr2. In plasmids, DnaK promotes initiation by reducing dimerization. A different mutation was in the 39-mer binding domain of RctB and inactivated it, indicating an alternative suppression mechanism. Paradoxically, although DnaK increases 39-mer binding, the increase was also achieved by inactivating the DnaK binding site of RctB. This result suggests that the site inhibits the 39-mer binding domain (via autoinhibition) when prevented from binding DnaK. Taken together, our results reveal an important feature of the transition from plasmid to chromosome: the Chr2 initiator retains the plasmid-like dimerization domain and its control by chaperones but uses the chaperones in an unprecedented way to control the inhibitory 39-mer binding.
Highlights
Replication of Vibrio cholerae chromosome 2 (Chr2) depends on molecular chaperone DnaK to facilitate binding of the initiator (RctB) to the replication origin
Participation of DnaK in 12-mer binding appears to follow the plasmid paradigm since we showed that RctB contains a dimerization domain which is folded to that of initiators of iteron plasmids
We initially determined interaction of DnaK with maltose binding protein (MBP)tagged RctB and untagged RctB by coimmunoprecipitation (Co-IP) and found that the MBP tag does not interfere with binding to DnaK
Summary
Replication of Vibrio cholerae chromosome 2 (Chr2) depends on molecular chaperone DnaK to facilitate binding of the initiator (RctB) to the replication origin. The initiation defect is suppressed by second-site mutations that increase 12-mer binding only marginally Instead, they reduce replication inhibitory mechanisms: RctB dimerization and 39-mer binding. The DnaK binding promotes replication inhibition by reducing an autoinhibitory activity of RctB. The same chaperones promote replication of a family of plasmids characterized by the presence of repeated initiator binding sites (iterons) in their origin, but they do so by a different mechanism. In addition to iteron binding, the chaperones promote plasmid replication by reducing the activity of an inhibitory regulatory mechanism called “handcuffing.” the dimers are inactive in iteron binding, they are believed to bridge monomer-bound iterons of sister origins (handcuffing), which inhibits further firing of the origins [9, 10]. The chaperones could facilitate replication both by promoting a positive function (initiator binding) and by reducing an inhibitory function (handcuffing)
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