Understanding supercoiling-dependent DNA recognition: a combined experimental and computational approach

Lead Research Organisation: John Innes Centre
Department Name: Biological Chemistry

Abstract

It is well known that DNA is the molecule of heredity and that the sequence of bases in DNA encodes the genetic information that defines an organism. DNA itself is generally organised in highly compacted structures, in particular supercoils in which the helical axis of DNA is coiled about itself. Supercoiling has a dramatic effect on the expression of genes and has been shown to act as a global regulator of gene expression. Our understanding of how supercoiling influences the behaviour of DNA is far from complete, particularly at the molecular level. Therefore we have developed a model system involving small DNA circles that we can analyse both by experimental methods and computer modelling. By combining these experimental and theoretical approaches we aim to understand how DNA recognition is affected by supercoiling, and how we can use supercoiling to influence biological processes.

Technical Summary

The strength and specificity of many DNA-protein interactions is sensitive to DNA supercoiling, to the extent that E. coli has been shown to re-programme its transcriptome by modulating the superhelical state of its genome. However, we still have little understanding of how such control mechanisms operate. Despite the numerous DNA and protein-DNA structures available in the Protein Database, which have shown the importance of DNA structure and flexibility in DNA recognition, there is almost no structural information on supercoiled DNA because this is more difficult to study using traditional methods such as crystallography or NMR. We will develop tools that allow supercoiling-dependent DNA recognition to be studied in a highly controllable way. We will then use these tools to understand the physical principles that underlie supercoiling-dependent DNA recognition, so that it becomes possible to predict supercoiling-dependency in genome-level studies. The methodology uses specially designed small DNA circles whose structure and superhelical state is both predictable and controllable. Physical insight will come from integrated molecular dynamics (MD) simulations. We will employ three tractable systems to test our methodology: 1) DNA minicircles - these have controllable sequences and levels of supercoiling, and are sufficiently small to be amenable to atomistic simulation. 2) DNA triplexes - we aim to measure how DNA triplex formation is affected by supercoiling the DNA circle and to use triplexes as agents to probe and perturb DNA structure. 3) Phage 434 repressor - this is a paradigm example of a genetic switch that is sensitive to changes in DNA supercoiling and topology, which we will use as a model system of DNA-protein interaction to study supercoiling-dependent recognition.

Planned Impact

Who will benefit? 1. Academic researchers in the areas of ligand-DNA and protein-DNA interactions, and scientists interested in systems biology, i.e. how does supercoiling influence global gene expression (see Academic Beneficiaries). 2. Pharmaceutical companies involved in antibacterial drug discovery will potentially benefit from a broader understanding of transcriptional control in bacteria. 3. Biotechnology companies who may be able to exploit the properties of small DNA circles revealed by this research. 4. The drug-design community who will benefit from the improved theoretical understanding of molecular recognition. 5. The general public in terms of gaining a better understanding of how DNA functions (through our outreach activities) and of the applications of high performance supercomputing. How will they benefit? a. Improved understanding of ligand-DNA and protein-DNA interactions and of molecular recognition in general. b. New ideas for drug discovery. c. New ideas for bioengineering and synthetic biology What will be done to ensure that they have the opportunity to benefit from this research? i. Publication in high-profile journals and presenting the findings at relevant conferences (such as Topo2011 and the annual Biophysical Society meeting). ii. Media involvement via JIC TOC Comms. iii. Collaborations with UK and overseas partners. iv. Investigation of exploitation potential via PBL. v. Establishment of BBSRC-CASE studentships based on work stimulated by this project. vi. Continued engagement with local schools.

Publications

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Bates AD (2013) Small DNA circles as probes of DNA topology. in Biochemical Society transactions

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Sutthibutpong T (2015) Comparison of molecular contours for measuring writhe in atomistic supercoiled DNA. in Journal of chemical theory and computation

 
Description We have set up a model system to understand the effect of supercoiling on the interaction of oligonucleotides and proteins with DNA. This has produced new understanding, of, for example, DNA triplex formation in small DNA circles and the effects of topology on these interactions. Essentially the advances have been methodological and will be utilised by us and others for new work.
Exploitation Route Methods for looking at the effects of supercoiling on DNA-associated processes and how to make small DNA circles. Atomistic simulation methods for supercoiled DNA. This work has provided the theoretical and experimental underpinning for looking at interactions of other molecules with DNA. This has led to new collaborations.
Sectors Pharmaceuticals and Medical Biotechnology

 
Description Ideas about making and using small DNA circles have been taken up by two companies: Twister and Inspiralis
First Year Of Impact 2014
Sector Pharmaceuticals and Medical Biotechnology
Impact Types Economic

 
Description Tackling tricky twists - how does DNA gyrase function inside living cells?
Amount £379,684 (GBP)
Funding ID BB/R001235/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 03/2018 
End 02/2021
 
Description Leake 
Organisation University of York
Country United Kingdom 
Sector Academic/University 
PI Contribution Provision of materials and expertise
Collaborator Contribution They are carrying out high-resolution microscopy experiments using material generated in my lab.
Impact One published paper
Start Year 2017
 
Description Pyne 
Organisation University College London
Department UCL Energy Institute
Country United Kingdom 
Sector Academic/University 
PI Contribution We have provided DNA minicircles for analysis
Collaborator Contribution She has been analysing DNA sample using atomic force microscopy.
Impact Paper in preparation
Start Year 2015