In order to understand human disease at a molecular level, we are attempting to determine the molecular structures of the key complexes involved in disease processes. To do this we take advantage of both x-ray crystallography and electron cryo-microscopy. Protein structure determination remains an extremely difficult process, frequently taking many years and fraught with difficulty, however great strides have been made recently, particularly in the field of electron cryo-microscopy, resulting in the award of this year’s Nobel Prize for Chemistry. The section remains at the forefront of the structural biology community, both in the UK, and worldwide.
Structures and Mechanisms of key components in Eukaryotic DNA Damage Signaling and Repair
Our genomic information is stored in the DNA. However, DNA is exposed to toxic chemicals, UV radiation, drug, alcohol and other metabolic products. Consequently tens of thousands of DNA bases are damaged each day in every human cell. Fortunately cells have developed sophisticated systems using multi-subunit macromolecular complexes to detect, process and repair these damages in a highly controlled and coordinated fashion. If this repair process is not working properly, such as when certain proteins are defective, the damaged DNA will not be repaired properly and/or promptly, and this can lead to changes in DNA, causing cancer or aging in the long run.
Several key events have been identified in this repair process. These include the detection and signaling of the damage, remodeling nucleosomes to expose the damaged DNA site and finally repair itself. We are currently using a multi-disciplinary approach to study many of the key macromolecular complexes involved in this process in order to provide a molecular understanding into these events. This knowledge can help us understand the cause of cancer and aging and can also provide new avenues for therapeutic development.
An evolutionary adaptation for the compaction of DNA in our cells – presents a natural barrier to cellular processes and growth. These barriers are regulated by chromatin remodelling enzymes, which act as gatekeepers to inaccessible regions of DNA. The human INO80 complex is one such gatekeeper and works with other molecular machines to facilitate the repair of genetic lesions that might otherwise lead to cancer and other diseases. Our lab is aiming to elucidate the molecular mechanisms of INO80 by combining biochemistry with state-of-the-art structural biology techniques. Some of our findings can be read about in recent publications in eLife (Willhoft et al., 2017) and Natural Structural and Molecular Biology (Aramayo et al., 2017).
Investigating protein complexes involved in DNA repair
The repair of double-strand DNA breaks is a vital process to the everyday survival of cells for every living organism. Multi-protein complexes with helicase and nuclease functions play a key role in preparing DNA breaks for repair. Studying the structure and function of such complexes continues to help develop our understanding of this crucial cell pathway, which may also help to understand how it fails in diseases such as cancers. Several functional insights have come from structures of the protein complexes AddAB, from Bacillus subtilis, and RecBCD, from Escherichia coli. The figure above shows the recent high-resolution cryo-EM structure of RecBCD bound to a DNA substrate mimicking a partially unwound double-strand break. The boxed figures show close-up views of the modelled structure showing the paths of each of the separate single strands of the DNA across the helicase domains of the complex. Bacterial helicase-nuclease complexes initially have a destructive function until they recognise the DNA to be its own and switch to initiate repair. As a result they play a role in the fight against invading pathogens such as viruses. Studying the mechanisms of how viruses evade these enzymes may help in the development of novel antibiotic treatments to help in the battle against the current global problem of antibiotic resistance.
Transcription, the synthesis of RNA from a DNA template, is an essential process to all living organisms and is carried out by RNA polymerase (RNAP). For this to occur, the double stranded DNA must be opened up and a template strand delivered into the RNAP active site. This process is called transcription initiation. Using cryo electron microscopy, we have recently determined structures of two complexes that show the early stages of transcription initiation by the bacterial RNAP bound to sigma54, a factor that binds to specific promoter elements but inhibits the spontaneous activation of transcription until acted upon by an activator protein utilising ATPase activities. These structures show how sigma54 acts to block the promoter DNA from entering the RNAP cleft that contains the active site (see figure) as well as its role in initiating distortions in the DNA that lead to the formation of the fully melted transcription bubble. In addition, we begin to distinguish the part played by the activator protein in the remodelling of the RNAP-sigma54 complex that is required for transcription activation.
Understanding SIgnal Integration in the (m)TORC1 Growth Pathway
The protein kinase Target Of Rapamycin (TOR) is a nexus for the regulation of eukaryotic cell growth. TOR assembles into one of two distinct signalling complexes, TOR complex 1 (TORC1) and TORC2 (mTORC1/2 in mammals), with a set of largely non-overlapping protein partners. (m)TORC1 activation occurs in response to a series of stimuli relevant to cell growth, including nutrient availability, growth factor signals and stress, and regulates much of the cell’s biosynthetic activity, from proteins to lipids, and recycling through autophagy. mTORC1 regulation is of great therapeutic significance, since in humans many of these signalling complexes, alongside subunits of mTORC1 itself, are implicated in a wide variety of pathophysiologies, including multiple types of cancer, neurological disorders, neurodegenerative diseases and metabolic disorders, including diabetes. We are currently using a multi-disciplinary approach toward towards a molecular understanding of growth signalling, and hopefully to eventually open up new avenues for therapeutics.