DNA distortion sequence recognition and the control of R-M gene expression

Bacteria use the so-called restriction-modification (R-M) system to protect themselves from invasion by foreign DNA (e.g. from bacterial viruses). The R-M system works by producing two enzymes. The first, a so-called methyltransferase (M), marks the bacterium's own DNA by adding methyl groups at strategic positions. The second enzyme, an endonuclease (R), then breaks down DNA that is not correctly marked. The R-M system thus provides a way to destroy foreign DNA selectively. However, if the timing of the production of the two enzymes is disturbed, the endonuclease will destroy the bacterium's own DNA, leading to the bacterial equivalent of an autoimmune disease and to death of the bacterium. To avoid this, so-called controller (C) proteins regulate the synthesis of the two enzymes by binding to the appropriate sites on the DNA that control the individual R-M genes. This leads to a complex regulatory network with positive and negative 'feedback' circuits. Our aim in this proposal is to understand exactly how subtle changes in the DNA sequence influence how strongly the controller protein binds to the control regions of the genes, and how this dictates the order in which they are switched on and off. Also, we want to know how the shape of the DNA is distorted to allow the proteins to interact with the double helix, and how this is involved in the synergy involved when proteins bind to adjacent sites on the DNA. Once we understand these things in molecular detail, it may be possible to design novel anti-bacterial drugs that are specific for different strains of bacteria.

R-M systems encode a restriction endonuclease and a DNA methyltransferase that must act in closely coordinated concert in order to protect bacteria against invasion by foreign DNA. We have worked out the overall mechanism of the genetic switch in Esp1396I, an R-M system with a 'classical' C-protein recognition sequence upstream of its own gene, and have solved the structure of the first protein-DNA complex for any C-protein system. In this structure, two protein dimers are cooperatively bound to the intact C/R promoter to form a repression complex. The current proposal seeks to build on the success of this work and expand it to investigate the structure and mechanism of a novel class of R-M controller proteins where 'action at a distance' appears to be involved. In the course of this project, we aim to solve the structures of the complexes of the C.Esp1396I controller protein with individual operator sequences, OL, OR and OM by X-ray crystallography, to measure the DNA binding affinities of the protein for these three sites, and to thereby understand how the precise DNA sequences of the operators governs the order of binding, and subsequently, the temporal regulation of the restriction(R) and modification (M) genes. In addition, we will attempt to determine the structural basis of the cooperativity between binding to the OL and OR operator sites. We will also pursue studies of a quite novel class of R-M controller protein, exemplified by Csp231I, in which there is a large spacing between operator sites, and where the DNA sequences are punctuated by numerous runs of oligo-A and oligo-T sequences, characteristic of DNA curvature. Using a combination of X-ray crystallography and a battery of biophysical techniques (including AUC, ITC, SPR, DSC), biochemical methods (e.g. DNA footprinting, transcription assays) and site-directed mutagenesis, we aim to gain a full understanding of the structure, function and mechanism of R-M gene regulation.

This research will have an impact on the structural biology community and more generally, on our understanding of the mechanisms of gene regulation in bacteria and of horizontal gene transfer in bacterial communities. In the longer term, the work will provide the detailed knowledge and understanding of these mechanisms that could pave the way for the development of novel anti-bacterial drugs, which are increasingly required as bacterial resistance to existing antibiotics represents a major health risk. In a broader sense, the research will benefit the wider community through outreach activities and international collaborations that will be pursued. Outreach to Schools and Colleges The University of Portsmouth runs a very successful outreach programme for local schools and colleges, called 'UP for It' (www.upforitclub.org.uk) that benefits directly from currently funded BBSRC research in our laboratory. As a department, we are leading engagement with local schools to foster an early link and to capture their scientific imagination. We provide mini-lectures in science and experimental demonstrations for junior school pupils. For example, we utilise our computer suite to teach magnification: from microscopes to synchrotrons. Each pupil is given a lesson in 3D molecular viewing software and, using our latest C-protein-DNA structure, they get to spin the molecules round, zoom in, and try to discover the differences between DNA and protein and how they 'stick' together. Even at this age, the children engage immediately with the computer software and have little problem with the concepts. We then have a quiz before they 'graduate', in full robes, with a certificate to take home. There is a more directed scheme for 11-16 year olds and sixth-formers, where they have dedicated laboratory visits. Classes are divided between our EM, NMR and X-ray crystallography facilities. In the latter, pupils are encouraged to mount C-protein crystals on our in-house X-ray generator and take diffraction images. The laboratory is arranged so that they can manipulate electron density maps and models on a large plasma screen. As part of their A-level assignment, they write reports based on the rational design of drugs, including antibiotics, in the context of structural biology techniques they have seen. These classes are very popular, with both pupils and teachers, and benefit greatly from the ability to demonstrate relevant newly published material. It is likely that this scheme will expand given that the UoP has invested in full time staff to coordinate these activities. The above activities are led by the co-applicant, Dr John McGeehan, who as a RCUK academic fellow is encouraged to engage with local schools and colleges. Media relations We have a well supported press office at the UoP and our work on R-M systems made the front page of the local newspaper. Following publication of results from the current BBSRC grant on the first C-protein-DNA complex (front cover of Nucleic Acids Research, July/Aug 2008), we had coverage from the regional press. A full page story was released in our regional newspaper on the use of our new X-ray facility in relation to C-proteins and the quest for novel antibiotics. The story was also taken up by our regional radio station where sections from an interview were aired every hour as part of the daily news program. Both media organisations are very keen to follow-up this story as new results are published. Collaborations In connection with the work in this proposal and closely related research, we have a number of international collaborations e.g. the Waksman Institute, Rutgers University, NJ, USA (Prof. Konstantin Severinov), the University of Erlangen, Germany (Prof. Tim Clarke, Prof. Andreas Burkovski), Leiden University Medical Centre, The Netherlands (Dr Raimond Ravelli), as well as exploitation of synchrotron facilities at ESRF Grenoble, and the Diamond Light Source, UK.