Single molecule investigations of bacterial DNA remodelling proteins

所有生物都面临着一项艰巨的任务，那就是必须压缩它们的DNA，以便它能够适应细胞内相对较小的空间。控制DNA压缩的过程也已知对于控制关键细胞过程（例如DNA复制和基因表达）是重要的。细菌通过几种机制实现这一点，包括通过一系列称为类核相关蛋白（NAP）的蛋白质的相互作用。除了压缩DNA之外，NAP被认为需要改变压缩的DNA分子的结构/重塑（称为重塑），以便参与重要细胞过程的其他分子可以访问DNA。对于许多细菌，特别是那些被称为革兰氏阳性细菌的细菌，这些蛋白质的作用以及它们如何与其他基本细胞过程联系在一起的了解非常少。然而，在最近的BBSRC资助的研究蛋白质被认为是参与DNA复制在革兰氏阳性生物体（B。枯草芽孢杆菌），我们已经发现两种蛋白质称为DnaD和DnaB具有新的DNA重塑活性。结合其他新的实验数据，我们的研究为这些蛋白质是DNA重塑和复制之间的联系提供了越来越多的证据。为了推进这些新的发现，我们现在需要显着提高我们对这些蛋白质的分子水平特性以及它们如何与DNA相互作用的知识。因此，该项目将采用显微镜方法，能够成像和记录DNA，蛋白质及其复合物的单个分子上和之间的力，以提供此类数据。这些研究不仅有助于提高我们对B.subtilis中DNA重构过程的理解，而且还有助于提高我们对许多其他相关生物中重构蛋白功能的理解。从长远来看，这些信息将影响我们对医学相关革兰氏阳性菌（包括芽孢杆菌、葡萄球菌、链球菌和梭菌）内DNA复制和基因调控/表达过程的认识，并可能导致识别新的抗菌治疗靶点。

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.