PAR recognition by PARP1 regulates DNA-dependent activities and independently stimulates catalytic activity of PARP1

  1. Waghela Deeksha
  2. Suman Abhishek
  3. Eerappa Rajakumara

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Abstract

Poly(ADP-ribosyl)ation is predominantly catalyzed by Poly(ADP-ribose) polymerase 1 (PARP1) in response to DNA damage, mediating the DNA repair process to maintain genomic integrity. Single strand (SSB) and double strand (DSB) DNA breaks are bonafide stimulators of PARP1 activity. However, PAR mediated PARP1 regulation remains unexplored. Here, we report ZnF3, BRCT and WGR, hitherto uncharacterized, as PAR reader domains of PARP1. Surprisingly, these domains recognize PARylated protein with a higher affinity compared to PAR but bind with weak or no affinity to DNA breaks as standalone domains. Conversely, ZnF1 and ZnF2 of PARP1 recognize DNA breaks but weakly to PAR. In addition, PAR reader domains, together, exhibit a synergy to recognize PAR or PARylated protein. Further competition binding studies suggest that PAR binding releases DNA from PARP1, and WGR domain facilitates the DNA release. Unexpectedly, PAR showed catalytic stimulation of PARP1 but hampers the DNA-dependent stimulation. Altogether, our work discovers dedicated high-affinity PAR reader domains of PARP1 and uncovers a novel mechanism of allosteric stimulation, but retardation of DNA-dependent activities of PARP1 by its catalytic product PAR. Therefore, our studies can be used as a model to understand the effect of one or more allosteric activators on the regulation of receptors or modular enzyme activities by another allosteric activator.

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  1. Review Commons

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    Reply to the reviewers

    Reviewer #1 (Evidence, reproducibility and clarity (Required)):

    In this manuscript the authors examine PAR-binding properties of PARP1 and identify ZnF3, BRCT and WGR domains as PAR-binding domains, which show cooperative effects on PAR binding. Their affinity for PARylated PARP2 was slightly higher compared to naked PAR. PAR binding competes with the binding to DNA strand breaks (SSB or DSB) and promotes DNA dissociation. PAR also reduces DNA-dependent activation of PARP1 catalytic activity. The findings are based on biochemical and biophysical experiments and the cellular significance of these findings has not been investigated.

    Major comments:

    1) The conclusion that the binding affinity of the three domains for PAR is high should be adjusted as the Kd is in the low micromolar range (3-5 uM). The PAR-binding affinity of individual domains compared to the full-length protein (Kd=39 nM) is thus rather low.

    Response: We agree with the reviewer that the Kd for PARP1 (measured using BLI) is low compared to that reported for individual PAR-binding domains (measured using ITC). But we have also measured the combined* KD* for three high-affinity PAR binding domains (ZnF3-BRCT-WGR) which is in the nanomolar range (~140 nM) which infers that the domains show cooperativity or synergy for PAR binding, while the affinity for PARP1 is ~39nM. The difference in KD can be attributed to the absence of ZnF1 and CAT domains in construct ZnF3-BRCT-WGR which could contribute to higher affinity in the case of PARP1.

    2) What is the affinity of PARP1 lacking ZnF3, BRCT and WGR for PAR? What is the DNA binding affinity of this mutant and its catalytic activity? If PAR competes with DNA binding, then this mutant is expected to show stronger DNA binding and stronger catalytic activation.

    Response: We thank the reviewer for raising the concern, but we differ from the reviewer’s assumption “If PAR competes with DNA binding, then this mutant is expected to show stronger DNA binding and stronger catalytic activation” since DNA recognition and DNA-dependent stimulation of PARP1 is independent of PAR binding.

    To address the concern, we cloned, expressed, and purified the ZnF1-2-CAT construct which lacks ZnF3, BRCT, and WGR domains (Figure S2j), and performed FP binding studies. Our results show that ZnF(1-2)-CAT binds to SSB with almost similar affinity as PARP1 while the affinity for DSB has reduced ~9 times due to lack of ZnF3 and WGR domain which contribute to DSB recognition (Figure S8a-d).

    We also assessed the catalytic activity of ZnF(1-2)-CAT using PNC1-OPT assay. Our results show that the construct could not be stimulated by SSB DNA but show a little more than basal-level activity, which is expected because the interdomain contacts required for communication of DNA-dependent stimulation signal from the N-terminus to the catalytic domain are lost due to the absence of ZnF3, BRCT, and WGR domains (Fig 6B). In addition, automodification (PARylation) domain, which is located between BRCT and WGR is also lost. We only performed the assay with SSB because it showed almost the same affinity as PARP1.

    3) The role of the WGR domain in DNA and PAR binding is unclear from the experiments in Figs. 4 and 5. The lower PAR concentration required to dissociate DNA from PARP1 in the case of full-length PARP1 vs ZnF-BRCT construct cannot be interpreted as being due to the WGR domain present in the full-length protein. To clearly show that this effect is due to the WGR domain, two experiments can be conducted: (i) compare full-length PARP1 and PARP1 mutant lacking WGR; (ii) compare ZnF-BRCT and ZnF-BRCT-WGR.

    Response: We thank the reviewer for suggesting experiments to further validate the role of the WGR domain in PAR-dependent DNA dissociation from PARP1. To perform the experiments, we cloned expressed and tried to purify ZnF(1-2-3)-BRCT-CAT (PARP1ΔWGR) and ZnF(1-2-3)-BRCT-WGR (PARP1ΔCAT) variants of PARP1 which lack the WGR domain and CAT domains (Figure S2l), respectively. We were unable to purify PARP1ΔWGR since it ended up in inclusion bodies.

    We conducted the FP binding experiments of ZnF(1-2-3)-BRCT-WGR with DNA breaks and it showed an almost similar binding affinity for DSB and SSB as that of PARP1 since all the domains involved in both the DNA breaks recognition are present in the construct (compare Figure 5c-d to Figure S8a-b). Furthermore, Ki values of PAR required to dissociate DSB and SSB from ZnF(1-2-3)-BRCT-WGR are ~ 1.8 and ~1.4 times, respectively, (Figure 5g-h) lesser than required for DNA dissociation from ZnF(1-2-3)-BRCT (Figure 5e-f), which again indicates that the WGR domain plays important role in PAR-induced DNA-break dissociation from PARP1.

    4) What is missing to make this study of higher impact are cellular assays to show, for example, how the absence of ZnF3, BRCT and WGR affects PARP1 recruitment to and retention at DNA damage sites.

    Response: We strongly agree with the reviewer that having cell-based experiments in the paper will give more insights into the PAR-dependent regulation of PARP1, but our data using several truncated and deleted variants of purified PARP1, binding, and competition binding studies, competition enzyme assays with multiple complementary techniques clearly shows that PAR plays major roles in modulating DNA dependent activities of PARP1. Certainly, this is in future plans with collaboration.

    Minor comments:

    The manuscript should be edited to improve readability and the presentation of the data.

    Response: We have edited the manuscript to improve the readability and presentation of diata.

    Reviewer #1 (Significance (Required)):

    Auto-PARylation of PARP1 was previously shown to cause its dissociation from DNA. Here the authors show that PAR binding through ZnF3, BRCT and WGR domains also causes dissociation from DNA and reduces PARP1 catalytic activity. These findings contribute to our understanding of how PARP1 DNA binding and activity can be regulated and will be of interest to researchers in the field of PARylation.

    I have expertise in biochemical analysis of PARylation.

    Reviewer #2 (Evidence, reproducibility and clarity (Required)):

    The authors investigate the impact of poly adenosine repeats on the parylation activity of Poly(ADP-ribose) polymerase 1 (PARP1). They show that PAR can bind to PARP1 through specific domains and that binding occurs in some cases to be as strong as binding to DNA. This work indicates that PAR binds to PARP1 sufficiently well to allosterically alter the biological consequences of PARP1 through parylation. For example, DNA appears to bind PARP1 and negatively regulate Parylation, therefore the work is potentially highly significant.

    **Referees cross-commenting**

    I agree with comments from Reviewer 1. Especially with the descriptions of binding affinity. low micromolar binding is relatively low affinity. The authors should revise this description. Other comments from Reviewer 1 are also appropriate.

    Response: We have addressed all the comments from reviewer 1.

    Reviewer #2 (Significance (Required)):

    General assessment: the authors use many purified domains from PARP1 that are purified and are used for quantitative binding experiments. The binding experiments appear to be done thoroughly with appropriate instrumentation.

    Advance: This work fills in a gap in understanding PARP1 and its key role in recruiting proteins to damaged DNA; that being the role of PAR in direct binding to PARP1.

    Audience: PARP1 is a major target for inhibition in treating cancer. The audience will include those interested in targeting PARP1 in a different way. As an enzymologist with interests in DNA repair, this paper was interesting and the results were properly analyzed.

    There are a few instances in which the text needs to be checked for grammar. Overall, the manuscript is clearly written. The data appear to be well presented except for items listed below.

    The equation used to fit fluorescence polarization data should be listed in the methods section. The competition binding studies were performed with 40 nM protein and 20 nM probe DNA. Under these conditions, Ki values below 20 nM should represent saturation binding rather than equilibrium binding. It would be useful to know whether the Ki values are reproduced with lower probe concentrations (below the Ki values). How is this taken into account in the data analysis?

    Response: Thanks for the reviewer suggestion to include equation to fit competition-binding data. As the reviewer suggested, we included the equation in the materials and methods section (Fluorescence Polarization (FP) studies).

    We completely agree with the reviewer that at a probe concentration of 20 nM, Ki values below 20 nM would represent saturation binding rather than equilibrium binding, but none of our Ki values are D * and *Ki *values differed marginally from corresponding values at 20 nM probe (DNA) concentration (Figure 4 and Figure S8a-b and e-h).

    Fig.4. The concentration of PARP1 and concentration of the DSB or SSB DNA should be stated. Also, the equation for fitting the data should be shown.

    Response: We have mentioned the concentrations of proteins and DNAs in the figure legends. The equation used for fitting competition-binding data has been included in the materials and methods (Fluorescence Polarization (FP) studies).

    Fig 5. list the concentration of enzyme and the 5-FAM DNA in the legend.

    Response: We have mentioned the concentrations of proteins and DNAs in the figure legends.

    Fig 6. In panel A, what form of DNA is shown in the gel image?

    Response: In Fig. 6, panel A, SSB has been used to show the DNA-dependent PARP1 stimulation. We have mentioned the name of DNA in figure, figure legend and corresponding text.

    Supplemental fig. 8 also need to list the concentration of the DNA.

    Response: We have mentioned the concentrations DNAs in the figure legends

    Reference is made to Figure S9, but there is no Figure S9.

    Response: Removed.

    A table that summarizes binding activity and catalytic activity would be helpful.

    __Response: __A table summarizing the binding affinity and catalytic activity of the constructs has been included in Supplementary File (Table S2).

  2. Review Commons

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

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    Referee #2

    Evidence, reproducibility and clarity

    The authors investigate the impact of poly adenosine repeats on the parylation activity of Poly(ADP-ribose) polymerase 1 (PARP1). They show that PAR can bind to PARP1 through specific domains and that binding occurs in some cases to be as strong as binding to DNA. This work indicates that PAR binds to PARP1 sufficiently well to allosterically alter the biological consequences of PARP1 through parylation. For example, DNA appears to bind PARP1 and negatively regulate Parylation, therefore the work is potentially highly significant.

    Referees cross-commenting

    I agree with comments from Reviewer 1. Especially with the descriptions of binding affinity. low micromolar binding is relatively low affinity. The authors should revise this description. Other comments from Reviewer 1 are also appropriate.

    Significance

    General assessment: the authors use many purified domains from PARP1 that are purified and are used for quantitative binding experiments. The binding experiments appear to be done thoroughly with appropriate instrumentation.

    Advance: This work fills in a gap in understanding PARP1 and its key role in recruiting proteins to damaged DNA; that being the role of PAR in direct binding to PARP1.

    Audience: PARP1 is a major target for inhibition in treating cancer. The audience will include those interested in targeting PARP1 in a different way. As an enzymologist with interests in DNA repair, this paper was interesting and the results were properly analyzed.

    There are a few instances in which the text needs to be checked for grammar. Overall, the manuscript is clearly written. The data appear to be well presented except for items listed below.

    The equation used to fit fluorescence polarization data should be listed in the methods section. The competition binding studies were performed with 40 nM protein and 20 nM probe DNA. Under these conditions, Ki values below 20 nM should represent saturation binding rather than equilibrium binding. It would be useful to know whether the Ki values are reproduced with lower probe concentrations (below the Ki values). How is this taken into account in the data analysis?

    Fig.4. The concentration of PARP1 and concentration of the DSB or SSB DNA should be stated. Also, the equation for fitting the data should be shown.

    Fig 5. list the concentration of enzyme and the 5-FAM DNA in the legend.

    Fig 6. In panel A, what form of DNA is shown in the gel image?

    Supplemental fig. 8 also need to list the concentration of the DNA.

    Reference is made to Figure S9, but there is no Figure S9.

    A table that summarizes binding activity and catalytic activity would be helpful.

  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

    In this manuscript the authors examine PAR-binding properties of PARP1 and identify ZnF3, BRCT and WGR domains as PAR-binding domains, which show cooperative effects on PAR binding. Their affinity for PARylated PARP2 was slightly higher compared to naked PAR. PAR binding competes with the binding to DNA strand breaks (SSB or DSB) and promotes DNA dissociation. PAR also reduces DNA-dependent activation of PARP1 catalytic activity. The findings are based on biochemical and biophysical experiments and the cellular significance of these findings has not been investigated.

    Major comments:

    1. The conclusion that the binding affinity of the three domains for PAR is high should be adjusted as the Kd is in the low micromolar range (3-5 uM). The PAR-binding affinity of individual domains compared to the full-length protein (Kd=39 nM) is thus rather low.
    2. What is the affinity of PARP1 lacking ZnF3, BRCT and WGR for PAR? What is the DNA binding affinity of this mutant and its catalytic activity? If PAR competes with DNA binding, then this mutant is expected to show stronger DNA binding and stronger catalytic activation.
    3. The role of the WGR domain in DNA and PAR binding is unclear from the experiments in Figs. 4 and 5. The lower PAR concentration required to dissociate DNA from PARP1 in the case of full-length PARP1 vs ZnF-BRCT construct cannot be interpreted as being due to the WGR domain present in the full-length protein. To clearly show that this effect is due to the WGR domain, two experiments can be conducted: (i) compare full-length PARP1 and PARP1 mutant lacking WGR; (ii) compare ZnF-BRCT and ZnF-BRCT-WGR.
    4. What is missing to make this study of higher impact are cellular assays to show, for example, how the absence of ZnF3, BRCT and WGR affects PARP1 recruitment to and retention at DNA damage sites.

    Minor comments:

    The manuscript should be edited to improve readability and the presentation of the data.

    Significance

    Auto-PARylation of PARP1 was previously shown to cause its dissociation from DNA. Here the authors show that PAR binding through ZnF3, BRCT and WGR domains also causes dissociation from DNA and reduces PARP1 catalytic activity. These findings contribute to our understanding of how PARP1 DNA binding and activity can be regulated and will be of interest to researchers in the field of PARylation.

    I have expertise in biochemical analysis of PARylation.

  4. Version published to 10.1101/2021.12.21.473685v2 on bioRxiv
  5. Version published to 10.1101/2021.12.21.473685v1 on bioRxiv