Structural and biochemical basis of interdependent FANCI‐FANCD2 ubiquitination

  1. Kimon Lemonidis
  2. Martin L Rennie
  3. Connor Arkinson
  4. Viduth K Chaugule
  5. Mairi Clarke
  6. James Streetley
  7. Helen Walden

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Abstract

Di‐monoubiquitination of the FANCI‐FANCD2 (ID2) complex is a central and crucial step for the repair of DNA interstrand crosslinks via the Fanconi anaemia pathway. While FANCD2 ubiquitination precedes FANCI ubiquitination, FANCD2 is also deubiquitinated at a faster rate than FANCI, which can result in a FANCI‐ubiquitinated ID2 complex (I Ub D2). Here, we present a 4.1 Å cryo‐EM structure of I Ub D2 complex bound to double‐stranded DNA. We show that this complex, like ID2 Ub and I Ub D2 Ub , is also in the closed ID2 conformation and clamps on DNA. The target lysine of FANCD2 (K561) becomes fully exposed in the I Ub D2‐DNA structure and is thus primed for ubiquitination. Similarly, FANCI's target lysine (K523) is also primed for ubiquitination in the ID2 Ub ‐DNA complex. The I Ub D2‐DNA complex exhibits deubiquitination resistance, conferred by the presence of DNA and FANCD2. ID2 Ub ‐DNA, on the other hand, can be efficiently deubiquitinated by USP1‐UAF1, unless further ubiquitination on FANCI occurs. Therefore, FANCI ubiquitination effectively maintains FANCD2 ubiquitination in two ways: it prevents excessive FANCD2 deubiquitination within an I Ub D2 Ub ‐DNA complex, and it enables re‐ubiquitination of FANCD2 within a transient, closed‐on‐DNA, I Ub D2 complex.

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  1. Version published to 10.15252/embj.2022111898
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    Referee #3

    Evidence, reproducibility and clarity

    Summary:

    Lemonidis et al perform a structural and biochemical investigation of FANCI-Ub/FANCD2 complex. The key findings are that FANCD2-Ub is more rapidly deubiquitinated that FANCI-Ub by USP1:UAF1, particularly in the context of the full FANCD2-Ub:FANCI-Ub complex. They present a structure of FANCI-Ub/FANCD2 and show key features that distinguish it from the other FA core complex structures. Several regions are unstructured in their model, which the authors propose is because of the absence of ubiquitin on FANCD2. The exposure of Ubiquitinated lysine of FANCD2 is suggested to provide a mechanism for the rapid reubiquitination of this site when bound to FANCI-Ub, and they also show this biochemically as a faster ubiquitination of FANCD2 when bound to FANCI-Ub instead of FANCI-Ub. A very neat conclusion is made about the dual role of FANCI-Ub in maintaining FANCD2-Ub in a DNA clamped state.

    Major comments:

    No major issues identified. The data supports the conclusions. Some mutagenesis studies that investigate the hypotheses generated by the structure would increase the overall impact of the study.

    Minor comments:

    The role of FANCI-Ub and DNA binding in protecting both FANCI and FANCD2 from deubiquitination was first shown in van Twest et al 2017 Molecular Cell, however this is not referred to in the manuscript.

    USP1:UAF1 has been shown to have DNA binding activity (eg Liang et al 2016 Cell Reports). Could this DNA binding activity potentially reduce its activity on I-Ub-D2-Ub in the presence of DNA (ie through sequestration away from the substrate by binding to other DNA?). This could be tested by showing that titrating the amount of DNA in the reaction does not inhibit deubiquitination. Also, one previous study showed that DNA stimulates deubiquitination of FANCD2. This study used complexes where FANCI was not ubiquitinated, but is a different result to what is seen in Figure 4A (but perhaps because deubiquitination is already 100% in the absence of DNA in these experiments). Some investigation/discussion of this discrepancy would be good to include. Some further discussions speculating why a FANCI-Ub is necessary in the FA pathway (ie why not just have FANCD2-Ub as the clamping mechanism given that it is sufficient to lock the protein onto DNA?) would increase the interpretation of the work by non-specialists.

    Significance

    The work provides a significant advance in our understanding of the mechanism by which di-monoubiquitinated FANCD2:FANCI clamps the complex onto DNA. The first structures of I-UbD2 are presented, and some evidence for why they exist in cells (as an intermediate state of deubiquitination of the complex). Previous studies already showed that FANCI-Ub and DNA binding prevent the deubiquitination activity (eg van Twest et al 2017, Arkinson et al 2018, Rennie et al 2020, Wang et al 2020) of USP1:UAF1.

    The work has clinical relevance - USP1:UAF1 inhibitors and FA core complex inhibitors are being developed by several biotech for cancer therapy (this is not referenced in the manuscript, and could perhaps be included in the introduction?). Researchers in several fields will be interested in the work: those working on FA pathway and mechanistic understanding of FANCD2 monoubiquitination, as well as those working more generally in ubiquitination and deubiquitination pathways. The structures included "complete the set" for all states of FANCD2:FANCI ubiquitination states, and this is a highly cited field. My expertise is in understanding the mechanistic basis of FANCD2 and FANCI ubiquitination and deubiquitination so I am well suited to evaluate all aspects of the proposal.

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    Referee #2

    Evidence, reproducibility and clarity

    The manuscript by Lemonidis et al describes a structural and biochemical basis for the interdependency of FANCI-FANCD2 monoubiquitylation. Continuing their landmark work in the FANCI-FANCD2-Ub Cryo-EM structure, this study shows that the homeostasis of FANCI-Ub-FANCD2-Ub (ID2-diUb) can be partly explained by accessibility differences of the ubiquitylated forms of FANCI and FANCD2 complex. Using structure and enzymatic assays, the authors show that FANCI-Ub is not as susceptible for deubiquitylation by USP1-UAF1 deubiquitinase (DUB) when in complex with FANCD2 and DNA. In contrast, FANCD2 deubiquitiylation is more efficient when FANCI is unmodified, and more protected when it is in complex with FANCI-Ub and DNA. Interestingly, FANCI ubiquitylation progresses at a faster rate when in complex with FANCD2-Ub and DNA. Even though this is somewhat an incremental study, nevertheless, the mechanisms of USP1 can or cannot remove ubiquitylated FANCI-FANCD2-DNA complex is intriguing and opens new areas of study to understand whether several different states of modified and unmodified FANCI-FANCD2 exists physiologically or not and what functional differences they may have.

    The major limitions of this study is the lack of functional connection to the described mechanisms of deubiquitylation or reubiquitylation of FANCI-FANCD2 complexes. In the future, hopefully, this group or others will generate point mutants that can selectively separate the functions of FANCI or FANCD2 deubiquitylation or FANCI or FANCD2 reubiquitylation to show the importance of dynamic regulation of these processes in a cellular or in vivo context.

    A major comment is whether the authors can by structural inference figure out what part of USP1 N-terminal extension is required for interaction and recognition of FANCI-FANCD2-Ub-DNA complexes. This is the only part of the study that wasn't very satisfying. Perhaps the authors can address this with some modeling and/or with additional mutational analyses on USP1.

    One minor comment is that the authors only cite the Smorgorzewska et al, Cell, 2007 paper for observing the first interdependency of FANCI-FANCD2 ubiquitylation in cells (Citations in the Intro and Discussion sections). The authors should also cite Sims et al NSMB, 2007 for the exact same observation shown in the two papers just to be consistent.

    Significance

    Significance of the study is as indicated above.

    The audience is the Fanconi Anemia and ubiquitin field.

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    Referee #1

    Evidence, reproducibility and clarity

    In this manuscript, Lemonidis et al concentrate on the role of FANCI ubiquitination in the ubiquitination and deubiquitination of the FANCD2 in the FANCI-FANCD2 complex. They work with purified FANCI and FANCD2 proteins that were in vitro ubiquitinated and with purified USP1-UAF1, allowing them to test the kinetics of the ubiquitination and deubiquitination reactions under different conditions. They also show a cryo EM structure of IUbD2 at a resolution of 4.1Å.

    Using biochemical assays, the authors show that ubiquitination of one of the subunits (FANCI or FANCD2) enhances the ubiquitination of the other. D2Ub can be de-ubiquitinated with high efficiency, with no protection by DNA and FANCI. However, when FANCI is ubiquitinated, FANCD2 de-ubiquitination is decreased. It is also more easily re-ubiquinated. The efficiency of de-ubiquitination of FANCIUb is strongly decreases when complexed to DNA and FANCD2, and it is independent of the FANCD2 ubiquitination. The structure shows that the IUbD2, like IUbD2Ub and ID2 Ub, is clamped on the DNA, but the FANCD2K561 site in exposed explaining how re-ubiquitination of the FANCD2 is facilitated.

    Major comments:

    The key conclusions are overall convincing. The IUbD2 structure shows a conformation in which the FANCD2 lysine is more easily accessible than in ID2 (3B). Biochemical assays support the claim that the ubiquitination of FANCI and FANCD2 promote the ubiquitination of their opposing subunit in ID2 (FANCD2, and FANCI, respectively). Furthermore, robust biochemical assays and structures in Figure 4 support the claim that the ubiquitin on FANCI is relatively more protected than the ubiquitin on FANCD2, of which the latter can be de-ubiquitinated with high efficiency.

    1. The major caveat is that these structures were generated using "engineered Ube2T and SpyCatcher-SpyTag", and not via the FA core complex. Recent structural work by the Passmore and Pavletich labs shows the intricate process of ID2 complex ubiquitination, and propose a sequential ubiquitination model that is tied closely to the unique structural properties of the FA core and the ID2 interface. Similarly, the biochemistry is done in the absence of the core complex which may change the kinetics of the ubiquitination and deubiquitination. So, although the biochemical experiments performed are robust, these caveats need to be explicitly noted in the manuscript.
    2. Ideally, the authors should also solve cryo EM structure of IUbD2Ub and compare this to 6VAE to show that their in vitro ID2 assembly leads to structures aligning with the FA-core complex ubiquitinated form of ID2.
    3. I would suggest including ubiquitinated FANCD2 in the PIFE experiments in Figure 2, to assess differential binding affinity of I and IUb. This would help serve as a control that FANCD2 ubiquitination also strengthens the ID2 complex binding the DNA.
    4. The authors claim that "ubiquitination of either of the two subunits of ID2 (FANCI or FANCD2) actually favors ubiquitination of the other subunit" (supported by biochemical and structural data in Figure 3). In Figure 3A-B, it is difficult to draw a conclusion on the "accessibility of the lysine residues" from these figures. Authors should include a measurement of the distances between the Lysines and the NTD of the opposite subunit. This would add a numeric measure to "accessibility".
    5. Wang et al, 2020 show that the FANCI K523 is only accessible for ubiquitination upon prior FANCD2 ubiquitination. However, the IUbD2 state could be formed by FANCD2-deubiquitination of IUbD2Ub. Figure 5 may need to changed to remove the arrow ID2 state to IUbD2.
    6. In Figure 3B, the structure of IUbD2Ub should be included to allow a proper comparison of the Lysine accessibility of IUbD2 and ID2Ub versus IUbD2Ub.

    Minor comments:

    1. Please include specific method of expressing, purifying and ubiquitinating FANCI and FANCD2. This method doesn't become clear by referencing previous papers, and these methods might have changed.
    2. The final paragraph needs to be reworded. The following sentence is very confusing "Of these two interfaces, one is absent in equivalent ubiquitin-FANCI interactions in ID2Ub-DNA structure (Wang et al, 2020)(Fig. 4D-E)." It does not come across that ubiquitin of FANCIUb lacks an interface with FANCD2.
    3. "Our structure indicates that this ubiquitin-FANCD2 interface ..." probably should be "Our structure indicates that the interface between FANCD2 and the ubiquitin of FANCIUb ..."
    4. I would suggest adding additional supplemental figures of Figure 3B including 1 extra angle for each structure. This would help gauge "Lysine accessibility".
    5. Please add PDB images in Figure 4D from the IUbD2Ub structure of both the interface between FANCD2 and ubiquitin of FANCIUb, and FANCI and the ubiquitin of FANCD2Ub. This is important to allow for the comparison of the different ubiquitin interfaces amongst the IUbD2Ub, ID2Ub, and IUbD2 structures

    References:

    Previous in vitro and in vivo data well referenced in introduction and discussion.

    Statistics:

    Satisfactory

    Comments on time and resources:

    Solving the cryo EM structure of IUbD2Ub is not a trivial ask, but the authors may already have it or may be inclined to do it since they already have access to purified IUb D2Ub and have optimized cryo EM conditions for IUbD2 in this paper. The remaining suggestions seems to be minor (repeat of biochemical experiment, measurement of pre-existing data, and minor changes to figures).

    Significance

    This work fits very well with the recent structural advances describing ubiquitination and de-ubiquitination of the Fanconi ID2 complex: Wang et al. 2020 and 2021, Alcon et al. 2020. These papers were groundbreaking in describing the mechanism in which FANCD2 and FANCI are ubiquitinated by the FA core complex. The same group (Rennie et al. 2021) solved a structure of ID2 bound to USP1-UAF1 which was also important for understanding of the Fanconi anemia pathway function. The current manuscript sheds light on the role of the Ubiquitinated FANCI in the function of the FANCID2 dimer.

    The manuscript will be of interest to the DNA repair, genome integrity, hereditary diseases and ubiquitin fields. This review was written by an investigator in the genome integrity and Fanconi anemia field.

  6. Version published to 10.1101/2022.04.07.487446v2 on bioRxiv
  7. Version published to 10.1101/2022.04.07.487446v1 on bioRxiv