Before cell division takes place, DNA replication is required to produce two faithful copies of the parental genome. During this process, a helicase enzyme separates the two DNA strands and two copies of a DNA polymerase enzyme synthesise new DNA on the template strands. These enzymatic activities are combined with a large number of accessory activities into a large multi-protein complex, the replisome, to ensure faithful copying of the two anti-parallel strands in the double helix.
In Escherichia coli, a single pair of replisomes is responsible for duplicating the ~4.6 Mbp circular genome. Recent data from our group has shown that core components of the replisome dynamically bind and unbind from the complex, raising the question: which protein factor provides stability and processivity to the complex?
This work aims to determine whether the DnaB helicase, as opposed to the DNA polymerase holo-enzyme, is the factor underlying replisome stability. By marrying single-molecule fluorescence, microfluidics, and protein purification, we have created methods capable of direct observation of single replisome components during reconstituted DNA replication events. With our single-molecule resolution, we probe DnaB helicase stoichiometry and exchange, and thus infer stability in the context of a fully reconstituted and functional replisome.