HPF1 and nucleosomes mediate a dramatic switch in activity of PARP1 from polymerase to hydrolase

  1. Johannes Rudolph
  2. Genevieve Roberts
  3. Uma M Muthurajan
  4. Karolin Luger

  • Curated by eLife

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    Evaluation Summary:

    This manuscript describes a set of biochemical studies on the substrate and reaction specificity of poly(ADP-ribose) polymerase 1 (PARP1), an important antineoplastic drug target and component of DNA damage response. The most significant finding is that histone PARylation factor (HPF1) binding to PARP1 causes a shift from primarily PARylation activity to that of hydrolytic activity, which offers new avenues for understanding and controlling PARP1. While some of the observed effects need a modest amount of further explanation, the findings described in this paper are of broad interest to readers in the fields of DNA damage response, chromatin structure regulation, and to researchers studying PARP1 and issues related to NAD+ metabolism.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

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Abstract

Poly(ADP-ribose) polymerase 1 (PARP1) is an important player in the response to DNA damage. Recently, Histone PARylation Factor (HPF1) was shown to be a critical modulator of the activity of PARP1 by facilitating PARylation of histones and redirecting the target amino acid specificity from acidic to serine residues. Here, we investigate the mechanism and specific consequences of HPF1-mediated PARylation using nucleosomes as both activators and substrates for PARP1. HPF1 provides that catalytic base Glu284 to substantially redirect PARylation by PARP1 such that the histones in nucleosomes become the primary recipients of PAR chains. Surprisingly, HPF1 partitions most of the reaction product to free ADP-ribose (ADPR), resulting in much shorter PAR chains compared to reactions in the absence of HPF1. This HPF1-mediated switch from polymerase to hydrolase has important implications for the PARP1-mediated response to DNA damage and raises interesting new questions about the role of intracellular ADPR and depletion of NAD + .

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  1. Version published to 10.7554/elife.65773 on eLife
  2. eLife

    Reviewer #3 (Public Review):

    The manuscript "HPF1 and nucleosomes mediate a dramatic switch in activity of PARP1 from polymerase to Hydrolase" by Rudolph et al. studies the effect of HPF1 on the steps of the catalytic reaction of PARP1. They use various PARP1 activators i.e. free DNA and varied forms of core nucleosomes to quantify reaction rates in the presence and absence of HPF1, using several assays. The main point of the manuscript is the observation that in the presence of HPF1, PARP1 is converted to an NAD+ hydrolase, which releases free ADPr, instead of its normal activity to produce ADPr polymers. The PARP1 hydrolase activity has been described previously, but they now show that HPF1 increases it substantially under the conditions that they tested. The authors also describe their independent identification of HPF1 residue E284 as a residue that is essential for Ser modification, confirming previous structural and biochemical work from Ivan Ahel's group. Although the assays are well performed and controlled and yield important quantitative information that was missing in the field, the main result of the hydrolase activity of PARP1 is hard to reconcile with current knowledge of HPF1 effects in cell-based experiments.

  3. eLife

    Reviewer #2 (Public Review):

    This enzymological analysis of the DNA-repair protein PARP1 in the presence and absence of its recently discovered regulator, HPF1, is a welcome contribution to the field that provides new data as well as introducing a valuable conceptual framework (seeing PARP1 as simultaneously catalysing 4 different reactions) and novel assays. Some of its conclusions - e.g. regarding the importance of residues Glu284 and Asp283 within HPF1 - are an independent validation of some of those from a recently published study but here they are reached with partially orthogonal means and supported by additional data (e.g. precisely quantified stability, binding, and catalytic parameters). Moreover, the study offers new insights, with the most interesting observation pointing to the prevalence of NAD+ hydrolysis to free ADP-ribose by PARP1 in the presence of HPF1. The technical aspects of the study including the design, number of repeats, data presentation and analysis, and the level of detail provided in the method section are adequate.

  4. eLife

    Response to Reviewer #1 (Public Review):

    1. The kcat enhancement from employing nucleosome substrates is exceedingly small, and probably will not ever be clearly correlated to a specific structural feature. However, more concerning is a possible uncontrolled variable when examining the nucleosome substrates. Specifically, the nucleosome substrates which yield a distinctly higher kcat (Table 1) are the larger, trivalent nucleosomes. It seems prudent to show that simply adding more potential binding sites, or perhaps just adding more protein itself is not causing these small increases in kcat (relative to DNA alone).

    All of these experiments were performed as a function of DNA concentration (see Fig. 2D). That is, the kcat was determined at saturating concentrations of DNA, which means that adding more DNA does not change (increase) the measured kcat. While the differences may appear subtle, they hold true consistently across all the free DNA vs. nucleosome comparisons and pass a statistical t-test, as was cited in the text for the p18mer vs. Nuc165 comparison (p<0.001; pg 3). We have added the p-values for the direct comparisons of p165mer DNA vs. Nuc165, and 621mer DNA vs. LE-Tri to the text on page 4. Also note, we observe this seem effect of nucleosomes being a better activator than p18mer in the HPLC assay (see Fig. 5C).

    1. Concerning the assignment of E284 of HPF1 as the catalytic base in the deprotonation of the Ser hydroxyl, I'm wondering if there might be a dynamical explanation for its role instead. E284A causes a significant decrease in the KD for HPF1 binding, and an elimination of the observed PARylation activity, suggesting that it may play an allosteric role. Also, we see from Table 2 that H303Q also produces a large reduction in the activity and large reduction in the KD; the standard error on the H303Q binding data is very large, but does suggest that some observations were quite low (similar to E284A). Additionally, H303Q almost eliminates enzymatic activity as well. Overall, this set of data gives me pause about certainty of the assignment of E284 as the catalytic base, as there may be a more complex origin of the loss of enzymatic activity.

    Although a role in protein dynamics is possible for Glu284, we believe this residue meets the classic definition of a catalytic base as measured by loss of activity without loss of binding (see Fig. 4C) or decrease in protein stability (see Table 2). These types of studies and determinations have much precedence in enzymology and we have added a new references (ref45). As stated in the text, the presence of Glu284 near the active site in Ahel’s structure is a nice confirmation of our result.

    Regarding the activity of the mutants: We apologize for the wrong values listed in Table 2 for E292A and H303A that were derived from rate values in an earlier iteration of the Table when we were still trying to fit straight lines through the “burst” curves. We have corrected these using the same 30 s endpoint value as used for the WT data. Thus, it is now evident that both of these mutants have robust activity toward histone PARylation. (The D283A and E284A still have no detectable transPARylation of histones in this analysis method.) Also, we note that the H303Q mutant of HPF1 binds identically to WT HPF1 (790 nM vs 676 nM); it is not lower (or tighter) as the reviewer indicates.

    1. It may be that the reason that there is no apparent PARylation at the standard carboxylate residue sites (in the presence of HPF1) is that they are forming transient ester bonds with the anomeric carbon, which are labile to hydrolysis. I feel that a better development of the treadmilling effect would enhance the paper (e.g., mutation of the orthodox carboxylate nucleophiles and examination of changes in HPF1-induced hydrolytic activity). I'm not sure that it can be quantitatively shown that the shorter PAR chains in the presence of HPF1 account for the pool of free ADPR.

    The reviewer appears to suggest that mono-ADP-ribosylation to Glu/Asp residues via the anomeric carbon could be the origin of the treadmilling effect. However, extensive characterization of these resulting ester linkages by the Jacobson group (ref41) indicate that these linkages are not labile to hydrolysis under standard physiological or assay conditions. These of linkages show high resiliency to hydrolysis except under very basic conditions (1M NaOH).

    In this system it is not possible to remove the “standard carboxylate residues”. The promiscuity of PARP1 towards different Glu/Asp (>100 sites total in PARP1 alone; see Anthony Leung’s website) highlights the problems of this approach since previous attempts to remove the three most prevalent PARylation sites yielded no significant change in the activity of PARP1, with some of the many other Glu/Asp residues presumably becoming the most favored sites (ref 18).

  5. eLife

    Reviewer #1 (Public Review):

    This manuscript describes a set of biochemical studies on the substrate and reaction specificity of PARP1, an important drug target and component of DNA damage response. The focus of the work is on the specific role of HPF1, and how PARP1's numerous activities are altered by complexation with it and with a variety of substrates. There are many important findings described in this paper, which will be of great interest to the researchers studying PARP1 and issues related to NAD+ metabolism. Perhaps the most significant finding is that HPF1 binding to PARP1 causes a shift from primarily PARylation activity to that of hydrolytic activity, yielding a large pool of free ADPR. The paper is very well written. Addressing the following issues would provide clarity.

    1. The kcat enhancement from employing nucleosome substrates is exceedingly small, and probably will not ever be clearly correlated to a specific structural feature. However, more concerning is a possible uncontrolled variable when examining the nucleosome substrates. Specifically, the nucleosome substrates which yield a distinctly higher kcat (Table 1) are the larger, trivalent nucleosomes. It seems prudent to show that simply adding more potential binding sites, or perhaps just adding more protein itself is not causing these small increases in kcat (relative to DNA alone).

    2. Concerning the assignment of E284 of HPF1 as the catalytic base in the deprotonation of the Ser hydroxyl, I'm wondering if there might be a dynamical explanation for its role instead. E284A causes a significant decrease in the KD for HPF1 binding, and an elimination of the observed PARylation activity, suggesting that it may play an allosteric role. Also, we see from Table 2 that H303Q also produces a large reduction in the activity and large reduction in the KD; the standard error on the H303Q binding data is very large, but does suggest that some observations were quite low (similar to E284A). Additionally, H303Q almost eliminates enzymatic activity as well. Overall, this set of data gives me pause about certainty of the assignment of E284 as the catalytic base, as there may be a more complex origin of the loss of enzymatic activity.

    3. It may be that the reason that there is no apparent PARylation at the standard carboxylate residue sites (in the presence of HPF1) is that they are forming transient ester bonds with the anomeric carbon, which are labile to hydrolysis. I feel that a better development of the treadmilling effect would enhance the paper (e.g., mutation of the orthodox carboxylate nucleophiles and examination of changes in HPF1-induced hydrolytic activity). I'm not sure that it can be quantitatively shown that the shorter PAR chains in the presence of HPF1 account for the pool of free ADPR.

  6. eLife

    Evaluation Summary:

    This manuscript describes a set of biochemical studies on the substrate and reaction specificity of poly(ADP-ribose) polymerase 1 (PARP1), an important antineoplastic drug target and component of DNA damage response. The most significant finding is that histone PARylation factor (HPF1) binding to PARP1 causes a shift from primarily PARylation activity to that of hydrolytic activity, which offers new avenues for understanding and controlling PARP1. While some of the observed effects need a modest amount of further explanation, the findings described in this paper are of broad interest to readers in the fields of DNA damage response, chromatin structure regulation, and to researchers studying PARP1 and issues related to NAD+ metabolism.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

  7. Version published to 10.1101/2020.12.18.423372v1 on bioRxiv