Interactions in DnaB-DciA complex in Bacterial DNA Replication

Markstein 102

In bacteria, there is an initiation process that occurs between several different proteins for DNA replication. One of these steps involves the loading of the helicase onto DNA by a specific protein called a loader. DciA is a potential loader of the DnaB helicase. We want to determine which sections of each protein bind each other to use as a basis for a preliminary structural model of the DnaB-DciA complex. Such a model would allow comparison of DnaB-DciA against canonical helicase-loader complexes. Bacterial transformations were done to insert the plasmids containing full length or truncated constructs of histagged-DciA and untagged DnaB. Recombinant proteins were expressed using IPTG-inducible promoters. Protein-protein interactions were assessed by nickel affinity chromatography to determine if histagged DciA co-eluted with untagged DnaB. Chromatography fractions were collected and ran on an SDS page gel. The N-terminal domain of DnaB was found to be dispensible for protein-protein interactions. The C-terminal domain of DciA (DciA-CTD) was found to bind strongly to a fragment of DnaB containing its C-terminal domain and adjacent linker helix (DnaB-LH+CTD). The N-terminal domain of DciA (DciA-NTD) was found to bind weakly to full length DnaB (DnaB-FL). Our observed strong interaction between DciA-CTD and DnaB-LH+CTD matches well with canonical helicase loaders. However, our observed weak interaction between DciA-NTD + DnaB-FL is absent from canonical loaders, and represents a novel helicase-loader interaction.