Single molecule investigations of bacterial DNA remodelling proteins

All organisms face the daunting task of having to compact their DNA so that it can be fitted into relatively small spaces within their cells. The processes controlling DNA compaction, are also known to be important for the control of crucial cellular processes, such as DNA replication and gene expression. Bacteria achieve this via several mechanisms, including via the interaction of a range of proteins termed nucleoid associated proteins (NAPs). In addition to compacting DNA, NAPs are thought to be required to change the structure of/reshape the compacted DNA molecule (known as remodelling), so that the other molecules involved in important cellular processes can access the DNA. For many bacteria, and in particular those known as gram positive bacteria, the roles of such proteins and how they are linked to other fundamental cellular processes is very poorly understood. However, in recent BBSRC funded studies of proteins thought to be involved in DNA replication within a gram positive organism (B. subtilis), we have discovered that two proteins called DnaD and DnaB have novel DNA remodelling activities. Together with other new experimental data, our studies are providing increasing evidence for these proteins being the link between DNA remodelling and replication. To progress these novel findings, we now need to significantly improve our knowledge of the molecular level properties of these proteins and how they interact with DNA. This project will therefore employ microscopy approaches that are able to image and record the forces on and between single molecules of DNA, proteins and their complexes, to provide such data. These proposed studies will not only help improve our understanding of DNA remodelling processes in B.subtilis, but should also improve our understanding of the functions of remodelling proteins in many other related organisms. In the long term, such information will impact on our knowledge of the processes underpinning DNA replication and gene regulation/expression within medically relevant Gram positive bacteria (including in Bacilli, Staphylococci, Streptococci and Clostridia), and could potentially lead to the identification new antibacterial therapeutic targets.

Prokaryotic organisms exploit a combination of mechanisms to compact their genomic DNA, so that it can be packaged into small intracellular spaces. These processes include the interaction of a range of nucleoid-associated proteins (NAPs), and remodelling of the highly campact/condensed DNA structure by NAPs is thought to be required to permit access of those proteins directly involved in replication and also other crucial processes including the regulation of gene expression. Whilst the properties and behaviours of NAPs are relatively well understood in E.coli (a gram negative organism), our knowledge of such proteins with gram positives is still incredibly limited. In recent collaborative studies we have however discovered that the DnaD and DnaB primosomal/replisomal proteins from B. subtilis (a gram positive organism) exhibit novel DNA remodelling activities, which we proposed were similar to those observed for the E.coli NAPs, H-NS and HU. Combined with recent gene expression and structural data, we are gaining increasing evidence that these proteins may act as NAPs to regulate globally replication, transcription and repair processes within gram positives. To progress this research we now need to obtain fundamental quantitative biophysical data to understand in detail how these proteins function and alter the structure of the DNA at the molecular level. This project aims to employ techniques such as atomic force microscopy (AFM) imaging and single molecule force spectroscopy measurements to obtain such data, supported by complementary biophysical techniques when needed. Such studies will not only impact on our understanding of remodelling processes within B.subtilis, but will also shed new insight onto their roles within many other organisms, including within medically relevant Gram positive organisms (Bacilli, Staphylococci, Streptococci and Clostridia). In the long term, this in turn may lead to the identification of novel antibacterial targets.