Structural biology of the cell-cycle and DNA damage response

Lead Research Organisation: The Francis Crick Institute

Abstract

Cells must respond to a plethora of changes that occur in their immediate environment. These responses ultimately require changes in the complement of protein molecules within the cell and these are effected by commensurate changes in how genes are expressed. This poses a number of significant problems since cells are defined by a continuous membrane through which the signals arriving from outside must be delivered or ‘transduced’. Once inside, these signals must be amplified and propagated to the nucleus where gene expression largely occurs. A major means by which such signalling events are elicited is through pathways and cascades of chemical modifications of proteins in the cell. One of the most common of these is the transfer of phosphate molecules onto specific amino-acids of target proteins, a process known as phosphorylation. These chemical events are brought about by a large family of protein enzymes called kinases that use the ubiquitous, energy-rich molecule ATP as a source of the phosphate ‘label’. We now know that there are more than 500 distinct kinases encoded in the human genome and it is clear that defects in the precision with which phosphorylation events occur are a primary cause of many cancers and other major illnesses. Phosphorylation most often results in a change in the 3D structure of the protein target. This may allow the phospho-protein to bind to a partner protein and pass along the signal, or prevent an unwanted interaction. We primarily use a method called X-ray crystallography to study the structural changes and interactions that occur as a result of phosphorylation, in atomic detail. By understanding these events, we may be able to determine why these processes run amok, and how we may design drugs to combat the devastating effects associated with these diseases.

Technical Summary

This work was supported by the Francis Crick Institute which receives its core funding from the UK Medical Research Council (FC001000), the Wellcome Trust (FC001000),and Cancer Research UK (FC001000)

Cellular responses to DNA damage are crucial for the maintenance of genomic integrity and complex surveillance and repair mechanisms have evolved to deal with a variety of DNA lesions. Of particular note are pathways involved in responses to double-stranded DNA breaks (DSBRs) since, if undetected or incorrectly repaired, these can lead to genomic rearrangements that are characteristic of cancer cells. Like many other signaling processes, the primary response to DNA damage involves phosphorylation cascades. Of central importance is a family of large serine/threonine kinases (STKs) that are related to the PI3-class of lipid kinases and which includes representatives in all eukaryotes. The human orthologue, ATM, is mutated in patients suffering from the disease ataxia telangiectasia (AT) that is characterised by neurodegenerative and immune disorders, extreme sensitivity to ionising radiation (IR) and cancer susceptibility. Many ATM substrates have been identified including Nbs1, Mdc1, Brca1, p53 and the two major DDR ‘transducer’ kinases, Chk1 and Chk2. These molecules are part of a web of interacting factors that combine to protect the cell against DNA damage and dysfunction in any one of them may result in a predisposition to cancer.
We are primarily focused on the molecular mechanisms by which phosphorylation regulates the assembly of DDR complexes through the activity of phospho-dependent binding modules such as forkhead-associated (FHA) and BRCT-repeat domains1. These are often found within more complex, modular proteins called ‘mediators’ that function as assembly platforms for myriad DDR effectors. In parallel, we are interested in the structure, regulation and interplay of DDR and cell-cycle kinases themselves. Ultimately we wish to build up a structural and biochemical picture of these ‘phospho-networks’ with a view to understanding how genetic aberrations lead to diseases such as cancer and how they may be targeted for therapeutic intervention. From a methodological viewpoint, structural analysis of any biological systems that have post-translational modifications at their core presents significant technical hurdles that we have begun to address using a variety of kinase and phosphatase co-expression strategies and expressed-protein phoshopeptide-ligation.

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