The ability to replicate is a key characteristic of all living organisms, allowing copies of the genome to passed on to subsequent generations. Critical to the replication process is the control of initiation that occurs at the origin. Where this process breaks down or becomes deregulated, catastrophic genomes rearrangements can occur that can cause, for example, cancer. Replication initiation in bacteria requires the sequence-specific assembly of an extensive filamentous nucleoprotein complex that causes DNA unwinding and which facilitates the loading of the replicative helicases. Somewhat surprisingly, the exact architecture of this complex is unclear and little is known about its dynamics.We will study this process using proteins and DNA from Bacillus subtilis, a model system that is representative of bacterial origins as a whole.
We will principally use a state-of-the-art single molecule methods, such as magnetic tweezers microscopy. These will allow us to directly follow in real time changes in DNA structure, namely assembly of the nucleoprotein filament and duplex DNA unwinding. We will address how the principal regulatory protein, DnaA, assembles on DNA, what the DnaA binding sites within the origin do and the role of ATP. In addition we will study the roles of accessory proteins that may stabilise the initiation superstructure, such as DnaD, DnaC-I and DnaB. Our ultimate goal is to define the minimal origin and protein components required for initiation. In quantifying the dynamics of the process, skills will also be gained in the analysis and interpretation of complex data.
Keywords: DNA replication, single molecule microscopy, enzyme, ATPase, AFM, fluorescence