Report
Teaser, summary, work performed and final results
Periodic Reporting for period 2 - ICLUb (Regulation of DNA interstrand crosslink repair by ubiquitin.)
Teaser
Human cells are constantly exposed to various forms of DNA damage, both during normal processes of cell division, and from environmental factors. Damage to DNA can lead to accumulation of mutations that can threaten the stability of the genetic material, and lead to cancers...
Summary
Human cells are constantly exposed to various forms of DNA damage, both during normal processes of cell division, and from environmental factors. Damage to DNA can lead to accumulation of mutations that can threaten the stability of the genetic material, and lead to cancers.
One of the most toxic and difficult to repair types of DNA damage is when 2 strands of DNA become chemically linked to each other in an interstrand crosslink (ICL). Repair of ICLs involves a pathway of more than 20 proteins which cooperate to detect, remove, and repair the ICL. This project addresses a very important problem, namely the molecular basis for activation of this important, but poorly understood DNA repair process controlled by factors defective in Fanconi Anaemia, the most common inherited form of anaemia in humans. The fact that this pathway is so poorly understood at a mechanistic level makes it a pressing problem in biomedical research. Although there are only a relatively small number of individuals who suffer from Fanconi Anaemia itself, this population is clearly desperate for progress in ameliorating the devastating symptoms and early mortality of FA. However, the FA pathway is also required for maintaining several stem cell compartments, for suppressing tumourigenesis, and is a major barrio to aldehyde-induced cell death and organ failure. This is because the ground-breaking work from the Patel lab at the LMB in Cambridge has shown that aldehydes, oxidised lipids, and formaldehyde all produce DNA lesions that are heavily dependent on components of the FA pathway for their removal. Finally, the FA pathway acts to remove the DNA ICLs that are caused by several types of cancer therapies, including mitomycin C, cisplatin, and ionising radiation. Therefore blocking the FA pathway could provide new ways to sensitise tumours, or potentially resensitise tumours that have become resistant, to such therapies.
The whole process of ICL repair is driven by a single signalling event – one molecule of ubiquitin is attached to a very specific target on the surface of FANCD2, one of the components of the FA pathway. This signal is then somehow recognised by unknown factor(s), and is subsequently removed. All three of these steps are absolutely required for the repair of this complex type of DNA damage.
Our overall objectives are to understand on the molecular level how this critical pathway required for repair of ICLs is regulated by this particular signal.
1. How is the signal generated?
2. How is the signal read/recognised?
3. How is the signal removed?
Understanding the FA pathway has broad implications to understanding ICL repair, genome instability, cancer and ageing, and alcohol-induced liver failure, and will provide a framework for therapeutic intervention.
Work performed
1. In order to understand how the signal is assembled, we have discovered an allosteric mechanism of site specific ubiquitination of FANCD2 by the FANCL and FANCT enzyme pair. This discovery has led to us generating a form of the enzymes that have significantly enhanced activity towards the target proteins, enabling us to generate large quantities of modified material, which was needed to facilitate the search for readers or binders of the signal, and to enable us to understand how the removal of the signal occurs.The results generated while addressing this have been prepared for publication (Chaugule et al., currently under revision for Nature Chemical Biology), or are already published (Chaugule et al., 2019, Methods in Enzymology).
2.Using this purified material, we have established a workflow for capturing proteins from cell lysates that have been treated with different DNA damaging agents, in an effort to identify binders of the monoubiquitin signal. We are also testing the hypothesis that the signal induces a conformational change in FANCD2 and FANCI that changes the affinity for different DNA structures. We are taking a cryo-elextron microscopy approach coupled with binding assays to explore this.
3. Also using the purified material generated while addressing the first point, we have discovered a specificity-determining element in USP1-UAF1, the enzyme responsible for removing the the signal from FANCD2. USP1-UAF1 has at least 3 substrates, all of which play a role in DNA damage repair, making this enzyme a very attractive target for cancer therapy development. By reconstituting each of these substrates and probing the molecular requirements for signal removal, we discovered an unexpected motif in USP1 that is responsible for targeting FANCD2 specifically, but not the other substrates.
4.In addition to the central aims of the work package, we have also used the reagents generated to explore the feasibility of targeting the FANCT/FANCL enzymes for small molecule or fragment binding. We have conducted a proof-of-principle fragment screen (Morreale et al., 2017a, J. Med. Chemistry), and unexpectedly uncovered a mobile secondary structure element in FANCT which in turn guided us towards the allosteric mechanism discovered in point 1 above (Morreale et al., 2017b, J. Med. Chemistry).
C. Arkinson, V.K.Chaugule, R. Toth & H. Walden Specificity for deubiquitination of monoubiquitinated FANCD2 is driven by the N-terminus of USP1 (2018) Life Science Alliance 12 October 2018. DOI: 10.26508/lsa.201800162
V.K.Chaugule, C. Arkinson, R. Toth & H. Walden An allosteric network in E2s drives specificity for RING E3 catalysed ubiquitin signals (2018) bioRxiv doi: https://doi.org/10.1101/429076 currently under review at Nature Chemical Biology
V.K. Chaugule, C. Arkinson, R. Toth & H. Walden Enzymatic Preparation of Monoubiquitinated FANCD2 and FANCI proteins (2019) Methods in Enzymology 618:73-104. doi: 10.1016/bs.mie.2018.12.021.
F.E. Morreale, A. Bortoluzzi, V.K. Chaugule, C. Arkinson, H. Walden* & A. Ciulli*. Allosteric targeting of the Fanconi anemia E2 Ube2T by fragment screening. Journal of Medicinal Chemistry (2017a) DOI: 10.1021/acs.jmedchem.7b00147
F.E. Morreale, A.Testa, V.K. Chaugule, A. Bortoluzzi, A. Ciulli & H. Walden. Mind the metal: a fragment library-derived zinc impurity binds the E2 ubiquitin-conjugating enzyme Ube2T and induces structural rearrangements. Journal of Medicinal Chemistry (2017b) DOI: 10.1021/acs.jmedchem.7b01071
Final results
As outlined above, we have already used some of our work in generating reagents to explore the feasibility of targeting the FA pathway. Our discovery that the FANCL/FANCT pair use an allosteric mechanism to drive the site-specific targeting of FANCD2 now gives us a scaffold to design small molecules capable of mimicking the allosteric regulation we have uncovered. Further results we expect to obtain include identification of a reader of the ubiquitin signal, a structural determination of the mechanism of signal removal, and the conformational change we anticipate in response to ubiquitin addition.