Periodic Reporting for period 5 - ICLUb (Regulation of DNA interstrand crosslink repair by ubiquitin.)
Período documentado: 2022-04-01 hasta 2022-08-31
One of the most toxic 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 pathway is called the Fanconi Anemia (FA) pathway, and mutations within it lead to Fanconi Anemia, a genome instability disorder that leads to bone marrow failure and high incidence of head and neck cancers. The FA pathway, like other DNA repair pathways, is regulated by highly-specific ubiquitin signals. Site-specific attachment of monoubiquitin to FANCD2 and FANCI, is accomplished by the E2/E3 ubiquitin-conjugating and ligase enzymes for the pathway. Importantly, not only is the monoubiquitination of FANCD2 and FANCI required for DNA repair, the signal must also be removed, by a dedicated deubiquitinase (DUB), USP1-UAF1.
Although Fanconi Anemia itself is rare, the FA pathway is also required for suppressing tumourigenesis, and is a major barrier to aldehyde-induced cell death and organ failure. It also 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. This signal is then somehow recognised by unknown factor(s), and is subsequently removed. All three of these steps are absolutely required for the DNA repair.
The objectives of the action are to understand on the molecular level how the FA pathway 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?
During this Action, we worked out the molecular basis for the very specific signal, and how it is attached to its target. We discovered that the reader of the signal was contained within the target itself, causing a conformational change that secures the targets to DNA. We determined the atomic structure of the proteins responsible for removal of the signal, including identifying that the removal is as specific as the assembly. Finally, we worked out how to block the removal of the signal, by understanding the mode of action of an inhibitor.
Although Fanconi Anemia itself is rare, the FA pathway is also required for suppressing tumourigenesis, and is a major barrier to aldehyde-induced cell death and organ failure. It also 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. This signal is then somehow recognised by unknown factor(s), and is subsequently removed. All three of these steps are absolutely required for the DNA repair.
The objectives of the action are to understand on the molecular level how the FA pathway 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?
During this Action, we worked out the molecular basis for the very specific signal, and how it is attached to its target. We discovered that the reader of the signal was contained within the target itself, causing a conformational change that secures the targets to DNA. We determined the atomic structure of the proteins responsible for removal of the signal, including identifying that the removal is as specific as the assembly. Finally, we worked out how to block the removal of the signal, by understanding the mode of action of an inhibitor.
1. How is the specific signal generated? There are >60 potential ubiquitination sites on the surface of FANCD2, but only one is specifically targeted by the E2/E3 pair, Ube2T/FANCL. We discovered an allosteric mechanism propagated across the surface of Ube2T that supports the activity of Ube2T. Our insights enabled us to engineer an enhanced version of Ube2T capable of faster and more complete ubiquitination of FANCD2, which proved invaluable for the subsequent aims of the project. In addition to the allostery, we worked out the molecular basis for the site specificity, and found it depended on a complementary arrangement of charged residues, similar to an ionic “cage” (Chaugule et al., 2020, Nature Chemical Biology;Chaugule et al., 2019, Methods in Enzymology).
2. How is the specific signal read? Many researchers have proposed that there is a recognition factor that reads the signal on FANCD2. Based on our engineered enzymes, we generated large quantities of modified FANCD2 and FANCI, and tested the hypotheses that the function of the modifications might not be for recruiting another protein, but rather support the DNA damage surveillance activity of FANCD2. We determined a structure of FANCD2-Ub in complex with FANCI, and discovered a conformational change that converts the ID2 heterodimer into a DNA “clamp”. We also found that modification of FANCI impedes the deubiquitination of FANCD2. We propose that the slowing of the deubiquitination of FANCD2 prolongs the signal on FANCD2 and enhances the DNA binding, perhaps stabilising the ID2 heterodimer at sites of ICLs to allow repair processes to take place (Rennie et al., 2020, EMBO Reports). We have also determined the structure of modified FANCI-Ub in complex with FANCD2, revealing that this signal is also sufficient to cause the conformational change to a DNA clamp, and favours modification of FANCD2 (Lemonidis et al., 2022, under revision at EMBO Journal, preprint on BioRxiv).
3. How is the signal removed? Removal of the signal is as important as assembly, yet it is much harder to study signal removal at the molecular level due to the prerequisite of having modified material to work with. Aim 1 resulted in high-quality substrates FANCD2-Ub and FANCI-Ub, and in addition, we also generated a further USP1-UAF1 substrate, ubiquitinated PCNA. Prior to our work, there was no structural insight into USP1-UAF1, and understanding of substrate targeting was poor. Whereas many USPs hydrolyse ubiquitin-ubiquitin linkages, USP1 targets ubiquitin-substrate conjugates at specific sites. We made many constructs of USP1 and reconstituted the deubiquitination of each substrate, and we found that the N-terminus of USP1 harbours a FANCD2-specific binding sequence required for deubiquitination of K561 on FANCD2. In contrast, the N-terminus is not required for direct PCNA or FANCI deubiquitination. This is the first evidence of discrimination between protein-ubiquitin linkages (rather than ubiquitin-ubiquitin linkages) and opens up the possibility of separating DNA repair pathways that are regulated by USP1-UAF1 (Arkinson et al., 2018, Life Science Alliance).
4. Following on from this we purified the 0.5MDa complex of USP1-UAF1-FANCD2-Ub-FANCI and determined the structure using cryo-electron microscopy. The model of USP1-UAF1 in complex with monoubiquitinated FANCI-FANCD2 highlights a highly orchestrated deubiquitination process, with USP1-UAF1 driving conformational changes in the substrate. An extensive interface between UAF1 and FANCI, confirmed by mutagenesis and biochemical assays, provides a molecular explanation for the requirement of both proteins, despite neither being directly involved in catalysis (Rennie et al., 2021, Nature Structural and Molecular Biology).
5. Drug discovery aspects have taken us beyond the original scope of the action (see also point 6) - we have determined the structure of the USP1-UAF1-FANCD2-Ub-FANCI complex bound to a known inhibitor of USP1 activity, whose mode of action was previously unknown. The inhibitor has a highly unusual mode of binding, and explains the specificity of the compound. This structure will form the basis for future work to target USP1 for inhibition (Rennie et al., 2022, accepted in Science Advances, preprint on BioRxiv).
6. 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).
Overview of results:
Achieved main objectives of the Action. Dissemination via academic conferences, seminar presentations, a press release.
2. How is the specific signal read? Many researchers have proposed that there is a recognition factor that reads the signal on FANCD2. Based on our engineered enzymes, we generated large quantities of modified FANCD2 and FANCI, and tested the hypotheses that the function of the modifications might not be for recruiting another protein, but rather support the DNA damage surveillance activity of FANCD2. We determined a structure of FANCD2-Ub in complex with FANCI, and discovered a conformational change that converts the ID2 heterodimer into a DNA “clamp”. We also found that modification of FANCI impedes the deubiquitination of FANCD2. We propose that the slowing of the deubiquitination of FANCD2 prolongs the signal on FANCD2 and enhances the DNA binding, perhaps stabilising the ID2 heterodimer at sites of ICLs to allow repair processes to take place (Rennie et al., 2020, EMBO Reports). We have also determined the structure of modified FANCI-Ub in complex with FANCD2, revealing that this signal is also sufficient to cause the conformational change to a DNA clamp, and favours modification of FANCD2 (Lemonidis et al., 2022, under revision at EMBO Journal, preprint on BioRxiv).
3. How is the signal removed? Removal of the signal is as important as assembly, yet it is much harder to study signal removal at the molecular level due to the prerequisite of having modified material to work with. Aim 1 resulted in high-quality substrates FANCD2-Ub and FANCI-Ub, and in addition, we also generated a further USP1-UAF1 substrate, ubiquitinated PCNA. Prior to our work, there was no structural insight into USP1-UAF1, and understanding of substrate targeting was poor. Whereas many USPs hydrolyse ubiquitin-ubiquitin linkages, USP1 targets ubiquitin-substrate conjugates at specific sites. We made many constructs of USP1 and reconstituted the deubiquitination of each substrate, and we found that the N-terminus of USP1 harbours a FANCD2-specific binding sequence required for deubiquitination of K561 on FANCD2. In contrast, the N-terminus is not required for direct PCNA or FANCI deubiquitination. This is the first evidence of discrimination between protein-ubiquitin linkages (rather than ubiquitin-ubiquitin linkages) and opens up the possibility of separating DNA repair pathways that are regulated by USP1-UAF1 (Arkinson et al., 2018, Life Science Alliance).
4. Following on from this we purified the 0.5MDa complex of USP1-UAF1-FANCD2-Ub-FANCI and determined the structure using cryo-electron microscopy. The model of USP1-UAF1 in complex with monoubiquitinated FANCI-FANCD2 highlights a highly orchestrated deubiquitination process, with USP1-UAF1 driving conformational changes in the substrate. An extensive interface between UAF1 and FANCI, confirmed by mutagenesis and biochemical assays, provides a molecular explanation for the requirement of both proteins, despite neither being directly involved in catalysis (Rennie et al., 2021, Nature Structural and Molecular Biology).
5. Drug discovery aspects have taken us beyond the original scope of the action (see also point 6) - we have determined the structure of the USP1-UAF1-FANCD2-Ub-FANCI complex bound to a known inhibitor of USP1 activity, whose mode of action was previously unknown. The inhibitor has a highly unusual mode of binding, and explains the specificity of the compound. This structure will form the basis for future work to target USP1 for inhibition (Rennie et al., 2022, accepted in Science Advances, preprint on BioRxiv).
6. 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).
Overview of results:
Achieved main objectives of the Action. Dissemination via academic conferences, seminar presentations, a press release.