Initiation of DNA replication in Bacillus subtilis

Reorganising the architecture of the bacterial genetic material is a complicated and dynamic process with crucial consequences to bacterial physiology. Significant reorganisation of the genetic material is required to set the scene for the start of DNA replication and also to assist the separation of the new genetic material from the old one. The molecular mechanisms behind DNA reorganisation and remodelling are not understood. We have recently discovered that two essential replication proteins known as DnaD and DnaB from the bacterium Bacillus subtilis exhibit significant opposing DNA-remodelling activities. In an effort to establish whether these remodelling activities are of direct relevance to DNA replication and to bacterial physiology we are now concentrating our efforts to reveal the molecular mechanisms that underpin these remodelling activities and also to study the effects of limiting these two proteins in the cell. We are aiming to solve the crystal structures of the two domains of DnaD and also of the full-length protein. Mutagenesis methods in parallel with the structural studies but also assisted by the forth-coming structures, together with supercoiling and genetic complementation experiments will be used to study the mechanisms of DNA remodelling and their significance to bacterial physiology. The findings of this study will be crucial if we are to undertand how the genetic material is reorganised in relation to DNA replication in a large class of bacteria known as gram positives. This class of bacteria contains important pathogens like Staphylococcus aureus (responsible for MRSA infections) and our findings will be of general applicability to the discovery of new antibiotic targets.

Remodelling of the bacterial nucleoid is a dynamic process closely associated with DNA replication. This remodelling mechanism and its close association with replication is poorly understood. With our recent work we have discovered substantial remodelling activities for two essential primosomal/replisomal proteins from Bacillus subtilis known as DnaD and DnaB. We are now seeking funding to continue our research efforts and elucidate the mechanism(s) and role(s) of these activities on the physiology of gram positive bacteria. We have designed a multi-disciplinary and complementary set of experiments, encompassing X-ray crystallography, gene complementation, atomic force microscopy and traditional mutagenesis and supercoiling experiments to achieve the two main aims described above; (i) to reveal the molecular details of the DnaD-mediated remodelling mechanism and (ii) to reveal the in vivo role of DnaD and/or DnaB on bacterial physiology. We have obtain considerable preliminary data in the form of diffracting crystals for the Nd and Cd of DnaD, topoI relaxation assays that reveal an untwisting activity for DnaD, a technical breakthrough with the isolation of whole nucleoids and are therefore in a strong position to deliver our aims and to contribute towards the understanding of an essential bacterial function for which we know precious little.