Regulation of DNA repair pathways by monoubiquitin signals

The information required for human life is encoded in our DNA, which is copied every time cells divide. It is crucial that DNA is copied accurately, as errors and mutations can be passed on to the next generations of cells, and can give rise to many different diseases, particularly cancers. The DNA in our cells is under constant threat from DNA-damaging agents. These include external sources such as UV from sunlight, tobacco, pollution, among many more, but DNA damage also happens during normal replication, metabolism, and other physiological processes. Humans have evolved multiple different pathways for repairing the many different types of DNA damage that can occur. Several inheritable diseases arise from mutations in these pathways including ataxia telangiectasia and Fanconi Anemia. These pathways are controlled by complex signal relays to recruit the many different proteins and enzymes required to keep DNA replication an accurate, high-fidelity process. One such signalling relay is the use of monoubiquitin signals, whereby a single molecule of ubiquitin is attached to a specific position on a protein that is required for recruitment and regulation of downstream repair factors.
In many cancers, tumour cells are replicating more quickly than non-cancerous cells. As such, cancer cells are vulnerable to DNA damage because of the potential to slow down or stop DNA replication. This vulnerability is exploited in medicine, with targeted DNA damage being a major form of chemotherapy and radiotherapy. However, such treatments also cause damage in other cells. Furthermore, chemotherapy encourages the rapidly dividing cells in tumours to find ways around the damage, which can lead to resistance. These mechanisms of resistance to drug treatments are not yet well understood. One possibility is that cancer cells recruit components from other repair pathways to circumvent the damage being caused by targeting one particular pathway. However, we do not yet have a full understanding of how different DNA repair pathways interact and interplay with each other.
Two of these repair pathways - one for allowing bypass of DNA damage sites, and one for fixing the damage when two strands of DNA become linked to each other - are regulated by common signals that are created by a unique set of proteins. The same enzyme is required to remove the signal, a step required for completion of the repair. We hypothesise that the shared features of these pathways underpin the interplay and cooperation between pathways. We aim to dissect and define the molecular details of the synergies and collaboration between pathways, defining unique elements that are specific, and generic elements that are common. We will take an integrated approach to testing this hypothesis, uncovering the atomic details of the molecules involved in order to understand how they function, and assessing the ability of individual pathways to impact on each other in cell lines. We'd like to use our insights into the molecular components to understand the basis of the interplay, how they influence each other, and whether that deep molecular understanding can be exploited to develop strategies to target cancer cells.

Monoubiquitin signals regulate many essential cellular activities, in particular DNA replication and repair processes. Multiple DNA repair pathways are controlled by highly specific single ubiquitin signals. These include the Fanconi Anemia (FA) pathway which repairs DNA interstrand crosslinks, and the Translesion Synthesis (TLS) pathway which permits replication past the site of damage. Although each pathway has distinct dedicated enzymes for assembling the modification, they share a common regulator which removes the signal. The deubiquitinase (DUB) Ubiquitin specific protease-1 (USP1) is dysregulated in several cancers. In conjunction with its activating co-factor, UAF1, it deubiquitinates FANCD2 and FANCI in the FA pathway, and the proliferating cell nuclear antigen (PCNA) in the TLS pathway. We recently identified mutations in USP1 that disrupt its activity towards FANCD2, but not FANCI and PCNA. This gives us a unique opportunity to uncouple the repair pathways and investigate the influence of each pathway on the other, and how perturbations might modulate the pathway responses to replication stress and DNA damaging agents. This is exciting because of the role of the DNA damage response as a target for anticancer therapy, but also as a mechanism for development of drug resistance. Understanding the molecular mechanisms of interplay between DNA repair pathways may provide new possibilities for targeted therapies.
We aim to address this with the following objectives: 1) Determine the molecular mechanism of deubiquitination of specific substrates by USP1-UAF1. 2) Define the molecular basis of the regulatory posttranslational modifications and DNA on substrate targetting. 3) Understand the functional consequences of signalling in the FA pathway on TLS and vice versa. These studies will show what is important for substrate recognition, signal removal, and how this might be regulated in individual targets.