PARP1 condensates differentially partition DNA repair proteins and enhance DNA ligation

  1. Christopher Chin Sang
  2. Gaelen Moore
  3. Maria Tereshchenko
  4. Michael L. Nosella
  5. Hongshan Zhang
  6. T. Reid Alderson
  7. Morgan Dasovich
  8. Anthony Leung
  9. Ilya J. Finkelstein
  10. Julie D. Forman-Kay
  11. Hyun O. Lee

Reviewed by

Read the full article

Listed in

Log in to save this article

Abstract

Poly(ADP-ribose) polymerase 1 (PARP1) is one of the first responders to DNA damage and plays crucial roles in recruiting DNA repair proteins through its activity – poly(ADP-ribosyl)ation (PARylation). The enrichment of DNA repair proteins at sites of DNA damage has been described as the formation of a biomolecular condensate. However, it is not understood how PARP1 and PARylation contribute to the formation and organization of DNA repair condensates. Using recombinant human PARP1 in vitro , we find that PARP1 readily forms viscous biomolecular condensates in a DNA-dependent manner and that this depends on its three zinc finger (ZnF) domains. PARylation enhances PARP1 condensation in a PAR chain-length dependent manner and increases the internal dynamics of PARP1 condensates. DNA and single-strand break repair proteins XRCC1, LigIII, Polβ, and FUS partition in PARP1 condensates, although in different patterns. While Polβ and FUS are both homogeneously mixed within PARP1 condensates, FUS enrichment is greatly enhanced upon PARylation whereas Polβ partitioning is not. XRCC1 and LigIII display an inhomogeneous organization within PARP1 condensates; their enrichment in these multiphase condensates is enhanced by PARylation. Functionally, PARP1 condensates concentrate short DNA fragments and facilitate compaction of long DNA and bridge DNA ends. Furthermore, the presence of PARP1 condensates significantly promotes DNA ligation upon PARylation. These findings provide insight into how PARP1 condensation and PARylation regulate the assembly and biochemical activities in DNA repair foci, which may inform on how PARPs function in other PAR-driven condensates.

Article activity feed

  1. Review Commons

    Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.

    Learn more at Review Commons

    Reply to the reviewers

    The authors do not wish to provide a response at this time.

  2. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #2

    Evidence, reproducibility and clarity

    Sang and colleagues present in vitro evidence for purified PARP1 forming condensates with DNA under certain conditions. They report the PARP1/DNA/NaCl concentration ranges under which these condensates form, and the impact of adding the PARP1 substrate NAD+ that allows poly(ADP-ribose) production. The results are presented clearly for the most part. One wonders if these in vitro conditions are really representative of DNA repair foci; however, the study adds new information that will be useful to the field. As noted below, there is some concern about the lack of reversibility of the condensates.

    • DNA curtains assay. The imaging buffer is listed as including 25 mM NaCl and 2 mM MgCl2. Were these experiments performed in conditions of higher ionic strength? The lack of a response to the addition of NAD+ is puzzling. It seems that the condensing of the DNA is not reversible. For the data in panel C, would the DNA return to extended form upon further flow of buffer only? The data give the impression that the assay conditions promote a one-way road to an irreversible state, and it is hard to see how this should be interpreted. A different single-molecule study indicated that PARP1 condensation of long DNA is reversible with NAD+ addition (PMID 34380612). The different outcomes should be discussed.
    • DNA ligase assay. Figure 5E,F. How can it be certain that the ligation is actually taking place in the condensates under these conditions? Can the Cy5 signal be shown? Is there a way to separate the condensates from the dilute phase, and then analyze the ligation state of the DNA? Also, the reactions could be performed under the same conditions (e.g. uM concentrations of LigIII and XRCC1) as presented in the rest of the figure.

    The discussion mentions the impact of HPF1 on PARP1 and that the research team has used HPF1 in the past. Is there a reason for excluding it from the current study? In particular in an effort to address the reversibility/mobility of the condensates?

    Supp. Figure 1F. It would be useful to convert the ng/uL concentrations to micromolar concentrations, perhaps in the legend for each nucleic acid. This would make the results easier to relate to the rest of the data that is generally listed in micromolar.

    Figure 6A. ZnF3, BRCT, and WGR domains of PARP1 also bind to DNA and should probably be included, as it could help explain why full-length PARP1 is needed for the most robust condensate formation.

    Supp. Figure 1G. The legend for this panel indicates absence or presence of NAD+, and that the DNA concentration is indicated, but this does not seem consistent with the figure panel.

    Results, first sentence. There is a missing parenthesis.

    Figure 2G,H. It would be useful to see the plot of the other replicates of the fusion experiments.

    page 3. "(data not shown)" Probably worth including.

    page 4. "(Supp. Fig. 3B and 3D)" Also Supp. Fig. 3C ?

    Referees cross-commenting

    Reviewer #1 comments are clearly stated and justified. There is good overlap in the feedback.

    Significance

    Strength: the study provides parameters for studying PARP1 condensate formation. Differential impact on repair factors is interesting.

    Limitation: the reconstitution is missing elements that could have a very big impact (e.g. nucleosomes).

  3. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #1

    Evidence, reproducibility and clarity

    PARP1 plays important roles in the recognition and repair of DNA damage, primarily by catalyzing the formation of PAR at DNA breaks, which assembles various repair factors and other PAR-binding proteins. The anionic PAR scaffold was previously proposed to induce condensation of phase separating proteins including FUS (PMIDs: 26317470, 26286827), and PARP1 was recently reported to form condensates at DNA breaks to promote nucleosome dynamics and DNA end synapsis (PMIDs: 38320550, 38215753). However, several aspects of PARP1-dependent repair condensate formation and how such condensates contribute functionally to repair have remained unclear. Here, Sang et al. show that purified PARP1 forms viscous droplets in vitro in a DNA binding-dependent manner, enhanced by PAR. Interestingly, the downstream DNA repair factors XRCC1, POLB, DNA Ligase III, and FUS co-assemble with PARP1 and damaged DNA, albeit with different enrichment patterns, resulting in multiphase condensates. Functionally, the authors not only confirm a DNA end bridging function by PARP1-mediated condensation, but also report enhanced DNA end ligation. Their in vitro experiments, which are state-of-the-art and are overall well controlled, despite lacking an in vivo counterpart, provide an important step forward in the reconstitution of multi-step DNA repair reactions in repair condensates.

    Major comments:

    1. In addition to the short oligonucleotides that were used in this study to evaluate DNA-dependent PARP1 condensation (Table S2), the use of circular plasmid DNA (nicked or broken to resemble SSBs or DSBs, respectively) should be considered to corroborate key findings.
    2. Although not essential for the main conclusions, it would be very interesting to address the role of PARG on PAR-dependent multiphase condensates. Based on the methods section, the authors have purified full-length PARG, so experiments to address the consequences of PARG-dependent PAR degradation on repair condensates and their disassembly seem feasible.
    3. If PAR increases the internal dynamics and mobility of PARP1 in condensates, why does it not seem to affect the DNA end bridging function?
    4. Catalytically inactive mutants of PARP1 could be employed to separate the PARP1-dependent DNA end bridging function from PAR-dependent modulation of PARP1 dynamics in condensates. Additionally, it would help to show that the DNA end bridging function depends on the ZnF domains and can be modulated by conditions that alter PARP1 condensation (see also point 6).
    5. The differential organization of XRCC1, LIG3, POLB, and FUS is intriguing, but the implications of this behavior remain unclear. Can the droplet assays be adapted to inform about the sequence of events from DNA damage recognition by PARP1 and PAR induction to the handing over of the break site to LIG3 for end ligation?
    6. The increase in end ligation, which correlates with PARP1- and PAR-dependent condensate formation, is very interesting. However, from the experimental setup it seems unclear if the observed effect is due to condensation or simply PARylation. Additional controls would be needed to substantiate a functional role of condensation for ligation (as implied also in the title of the manuscript). Perhaps it is feasible to modulate condensation (e.g. enhanced by crowding agents, reduced by salt or by 1,6-hexanediol) without affecting PARylation, and then reassess how this affects ligation.

    Minor comments:

    1. Please double-check if previous studies reaching similar conclusions are referenced appropriately.
    2. Please carefully double-check if all references to figure panels are correct.
    3. Please carefully double-check if the methods descriptions and discussion match the displayed experimental procedures and results.
    4. Supplemental Figure 3 seems to contain only negative results. Consider showing experimental conditions with the same proteins or protein combinations that result in droplet formation as well.

    Referees cross-commenting

    The comments by reviewer #2 seem comprehensible and justified. Similar points are addressed, e.g. major points 2, 3, and 6 in review #1.

    Significance

    Several new and exciting findings on PARP1-dependent and PAR-modulated repair condensate formation are presented, including the multiphase behavior and the functional contribution to DNA compaction, end synapsis, and ligation. The study extends previous work on PARP1/PAR-triggered liquid demixing and complements very new and recently published work by the Alberti and Kay labs on PARP1 condensation at DSBs. The study makes an important step forward towards the reconstitution of DNA repair reactions inside multi-component repair condensates in vitro, which may eventually allow making testable predictions for repair condensate functions in vivo. Strengths of the current study include complementary state-of-the-art in vitro techniques such as biochemical assays, multi-component droplet assays, and single molecule experiments, which were conceived and conducted carefully. Limitations relate primarily to the undemonstrated relevance in cells, which may, however, be beyond the scope of the current study. A broad audience of basic researchers in the areas of genome stability and biomolecular condensates will likely be interested in this study.

  4. Version published to 10.1101/2024.01.20.575817v1 on bioRxiv