The Role of DNA dynamics in damage recognition

The ability of cells to faithfully replicate and transmit their genetic information (their DNA) is essential in all living organisms. The process requires accurately reproducing a sequence of billions of nucleotide units into two identical copies. In addition, natural cellular processes and environmental factors can result in damage to the DNA that must be repaired. Failing to precisely duplicate and/or repair the genetic material can result in diseases including cancer. During DNA replication, DNA structures deviating from the double helix are formed that must be processed by specialized enzymes to return the DNA to its normal double helical state. Another class of enzymes repair chemically damaged DNA. In this project we want understand how these different classes of enzymes recognize aberrant DNA within a vast sea of normal DNA.This is the very first step in the repair process, and as such is of central importance.

Many people have looked at this process from the point of view of the proteins, but we will seek to discover if differences in the dynamics of the DNA are also important.
Specifically, we will discover if changes in DNA flexibility could be one mechanism by which repair enzymes quickly locate their substrates.

To do this, we will look at the conformations (shapes) of individual DNA molecules, one at a time (and so overcome any ensemble averaging). We will also implement a new method, X-ray scattering interferometry technique (that has not yet been used in the UK) which works by measuring the distances between tiny gold crystals attached to the DNAs. This measurement is so fast that it provides a snapshot of all the different shapes a DNA adopts in solution i.e. the un-averaged, conformational ensemble. We will compare these conformational ensembles for normal duplexes and a range of aberrant DNAs, to understand the role of flexibility in genome stability.

In human cells, DNA is packed around proteins (histones), into particles called nucleosomes. In the last part of the project we will see if aberrant DNA structures pack differently to normal duplex DNAs, possibly leaving themselves more open for repair proteins to interact with.
Our project will use cutting-edge experimental methods, combined with computer simulations to understand the role of conformational dynamics in aberrant DNA recognition.

DNA is most often in its double helical form in the cell, but even under normal conditions, aberrant DNA structures are formed. These structures can be grouped into two classes: Class I is characterized by single-strand breaks in the DNA duplex, for example nicked, gapped and flapped DNAs, which are necessary intermediates produced during DNA replication and repair; Class II involves chemical modifications to individual DNA bases, for example alkylation, oxidation and deamination. Such aberrant DNA structures need to be processed to return the DNA to its normal duplex state, and damaged bases removed or repaired to maintain genomic stability.

In this proposal we will use cutting-edge biophysical approaches to address a fundamental biological question: Is there a common first step in aberrant DNA recognition by DNA repair enzymes?

Our hypothesis is that changes in flexibility of aberrant DNAs are exploited by DNA repair enzymes to rapidly locate their sites of action. We will test this through a comprehensive characterization of the conformational ensembles adopted by aberrant DNAs. These will be determined using a combination of single-molecule FRET measurements (which overcome ensemble averaging), and X-ray Scattering Interferometry with gold-nano-crystal labelled DNAs (which reports the non-time-averaged distribution of conformations). We will integrate our extensive experimental data with molecular simulations using maximum entropy and Bayesian approaches, to obtain conformational ensembles of different aberrant DNAs at atomistic resolution. Finally we will explore how the conformational dynamics of damaged DNA within the nucleosome core particle, to test if damaged DNA effectively makes itself more available for repair, due to inherent differences in wrapping dynamics.

Pharmaceutical companies: DNA damage response proteins are potential oncology targets. More broadly, the methodologies established here for determining the conformational ensembles of biomolecules are also of interest as inhibiting key enzyme motions has proved a successful strategy for some drugs. The PI is collaborating with Evotec who have a number of interests in this space. The UoS Research and Consultancy unit has negotiated contracts that protect our ability to publish any collaborative research involving Evotec, and any IP arising out of the project remains with UoS.

The PDRA: The most immediate impact will be on the PDRA employed on this grant. Given the interdisciplinary nature of the work, there are many training opportunities, as it is unlikely any PDRA will already have skills across all the areas covered in the project. The PDRA will benefit from learning to work with multiple collaborators in industry (Evotec), at national facilities (Diamond Light Source), and internationally (University of Copenhagen). This will allow the PDRA to build an excellent network of key contacts, in addition to the variety of skills both explicitly scientific and more general skills (communication, presentation).

Other Academic Groups: Our study will also impact the wider UK science community, as we will make available our cutting-edge methods which will be broadly applicable to many DNA and protein related research areas.