DNA damage is a frequent occurrence in cells and must be continuously monitored and repaired in order to maintain genome stability (Peterson and Côté, 2004). The abundant nuclear proteins poly(ADP-ribose) polymerase 1 and 2 (PARP1 and PARP2) serve as early detectors of DNA damage (Gibson and Kraus, 2012; Ray Chaudhuri and Nussenzweig, 2017). PARP1, upon binding to damaged DNA, binds NAD⁺ and transfers the ADP-ribose (ADPR) portion onto itself and other proteins. PARP1 then elongates this ADPR to form a chain in a process termed poly(ADP-ribosyl)ation (PARylation), which can take place on PARP1 (autoPARylation) or other proteins (transPARylation) (Gibson and Kraus, 2012; Morales et al., 2014; Bai, 2015). These PAR chains are attached to thousands of nuclear proteins with histones being the most abundantly PARylated proteins apart from PARP1 (Bonfiglio et al., 2017a; Larsen et al., 2018). More recently it has been shown that histone-linked PARylation depends on Histone PARylation Factor 1 (HPF1) (Gibbs-Seymour et al., 2016). Interestingly, HPF1 not only promotes transPARylation of histones but also alters the specificity of the modified residues from aspartate or glutamate to serine (Palazzo et al., 2018). The PAR chains attached to PARP1 and other proteins lead to the recruitment of DNA repair factors that contain PAR-binding motifs, such as macrodomains, WWE domains, or PAR-binding zinc fingers (Teloni and Altmeyer, 2016). It is thought that different types of PAR chains (i.e., long vs. short, branched vs. unbranched) may lead to recruitment of different repair factors in a concept termed the PAR code (Aberle et al., 2020; Karlberg et al., 2013). Deletion of PARP1 leads to increased carcinogenesis, and deletion of both PARP1 and its most closely related homolog, PARP2, is embryonically lethal (Amé et al., 1999; Ménissier de Murcia et al., 2003). PARP1 (and PARP2) are important targets for cancer therapy with now four clinically approved compounds (olaparib, rucaparib, niraparib, and talazoparib) in use for treatment of breast and ovarian cancer (Yi et al., 2019). Additionally, these and other inhibitors of PARP are being investigated as cancer treatments in combination with agents that damage DNA (e.g., temozolomide, cis-platinum, checkpoint inhibitors) (Yi et al., 2019). All clinically relevant inhibitors of PARP bind in the NAD⁺ binding pocket of the catalytic domain of PARP1 and PARP2 (Thorsell et al., 2017).

From a mechanistic point of view, PARP1 is a very complicated enzyme that catalyzes four different reactions (Alemasova and Lavrik, 2019). In the first reaction, NAD⁺ serves as a substrate for ADP-ribosylation of an amino acid side chain wherein the nucleophilic side chain attacks C1′′ to release nicotinamide (Figure 1A). A number of aspartates and glutamates in the BRCT domain of PARP1 itself are the primary sites of modification in autoPARylation reactions (Tao et al., 2009; Zhang et al., 2013; Vyas et al., 2014; Gagné et al., 2015). The lack of specificity for a particular site (or protein) has been frustrating and confusing the field for decades (Tao et al., 2009; Suskiewicz et al., 2020a). In the presence of HPF1, the specificity of PARP1 switches to serine side chains, again located primarily in the automodification regions between the end of the BRCT domain and the WGR domain for autoPARylation, and in histone tails for transPARylation (Larsen et al., 2018; Bonfiglio et al., 2017b). In the second reaction catalyzed by PARP1, NAD⁺ is used to extend the initially attached ADPR, wherein the 2′-OH (the acceptor hydroxyl) of this ADPR is deprotonated by Glu988 (Ruf et al., 1998; Figure 1B). Repeating this process, PARP1 builds long PAR chains of up to 200 units on itself with a large range reported for average chain lengths (Kawaichi et al., 1981; Sugimura and Miwa, 1982; Alvarez-Gonzalez and Jacobson, 1987), in competition with PARGs (Pascal and Ellenberger, 2015). In a third reaction, PARP1 uses NAD⁺ to add ADPR to create branch points by generating linkages using the 2′′-OH instead of the 2′-OH (Figure 1A). Branching has been shown to occur about once every 5–20 residues for autoPARylation (Alvarez-Gonzalez and Jacobson, 1987; Miwa et al., 1979) and is mechanistically feasible because of the pseudo-symmetry of ADPR that allows for binding of the elongating polymer in the opposite direction (Ruf et al., 1998). In the fourth and final reaction, PARP1 hydrolyzes NAD⁺ to generate free ADPR. This ‘treadmilling’ reaction becomes more dominant at extended reaction times and low concentrations of NAD⁺ (Bauer et al., 1986; Desmarais et al., 1991). We address the mechanism by which HPF1 affects initiation, elongation, and treadmilling in this article.

Figure 1

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In our approach, we build on the seminal experiments performed by Ahel’s group, who discovered HPF1 and validated its effect both in vitro and in vivo (Bonfiglio et al., 2017a; Gibbs-Seymour et al., 2016; Suskiewicz et al., 2020b). As we are particularly interested in studying how HPF1 mediates PARylation of histones, proteins that do not exist in free form in the cell, we use nucleosomes as both substrates and activators of PARP1. We have previously shown that nucleosomes bind tightly to PARP1 (1–13 nM) (Clark et al., 2012; Muthurajan et al., 2014). Of special relevance to both nucleosomes and HPF1, we have recently shown that HPF1 binds fivefold more tightly to a PARP1–nucleosome complex than to PARP1 bound to a DNA oligomer (Rudolph et al., 2021). We show that HPF1 not only changes the specificity of PARP1 from autoPARylation and glutamate modification towards transPARylation and serine modification, it also converts PARP1 into a strong NAD⁺ hydrolase, resulting in much shorter PAR chains and high concentrations of free ADPR. Our results have important implications for the cellular role of HPF1 in PARylation, the PAR code, and the DNA damage response.

We began our work by demonstrating that nucleosomes are better activators of PARP1 than free DNA (Figure 2, Table 1). Because the tested nucleosomes all activate equally well (regardless of overhang length, or mono- vs. tri-nucleosomes), our results extend previous observations about the promiscuity of PARP1 with respect to DNA activators (Langelier et al., 2014; D'Silva et al., 1999; Liu et al., 2017) using a more physiologically relevant activator. Based on the seminal work by Pascal and Black demonstrating that DNA activation of PARP1 is mediated by a series of conformational changes that disorder the HD domain and thereby open up the active site for binding of NAD⁺ (Dawicki-McKenna et al., 2015; Steffen et al., 2016), we hypothesize that nucleosomes are in some way more efficient in this process. Perhaps there are direct interactions between the unfolded (or alternatively folded) HD domain and nucleosomes that stabilize the open conformation and prevent this domain from collapsing back onto the catalytic domain. In fact, just such an interaction is observed by the loop in the HD domain of PARP2 (Leu302 and Arg303) with the linker DNA from the nucleosome (Bilokapic et al., 2020). Ongoing structural efforts will hopefully show whether this or some other interaction exists for PARP1, leading to the enhanced activation of PARP1 by nucleosomes vs. free DNA.

Upon adding HPF1 to assays using nucleosomes as both activators and substrates of PARP1, we show that the activity of PARP1 is significantly redirected towards histones and away from PARP1 (Figure 3, Table 1). Our results are consistent with those from the Ahel group obtained with free DNA and histone-derived peptides (Gibbs-Seymour et al., 2016) as well as from the Muir group using nucleosomes (Liszczak et al., 2018), but provide quantitative information regarding the extent of this switch. For example, HPF1 causes transPARylation of histones to be preferred over autoPARylation of PARP1 minimally by factors of 2–4 (Figure 3C, Table 2), a switch that is particularly dramatic as we observe no histone PARylation in the absence of HPF1 (Figure 3A). This switch in specificity is notable as autoPARylation is a simple one-body problem wherein PARP1 modifies itself, whereas transPARylation requires the correct interactions between PARP1, HPF1, and the histone tails in the nucleosomes. Also, it should be re-emphasized that, in addition to the switch towards transPARylation, HPF1 mediates the switch in amino acid specificity away from aspartate/glutamate towards serine, even on PARP1 itself (Figure 3D).

In an independent and unbiased approach to better understand the mechanism of how HPF1 effects the switch in amino acid specificity from glutamates and aspartates to serines, we have identified Glu284 as the catalytic base on HPF1 (Figure 4, Table 2), as also recently published by the Ahel (Suskiewicz et al., 2020b) and Yun groups (Sun et al., 2021). Importantly, we demonstrate that the E284A mutant has the same stability as wild-type HPF1 and binds even more tightly to the PARP1–nucleosome complex than wild-type HPF1. The occurrence of a shared active site formed by PARP1 and HPF1 is a completely unexpected and novel finding in the field of post-translational modification (Suskiewicz et al., 2020a). The PARP1–HPF1 complex has two catalytic bases. Glu284 on HPF1 mediates the deprotonation and activation of serine residues on acceptor proteins for the initiation reaction (Figure 4A), while Glu988 on PARP1 mediates the deprotonation and activation of the 2′- or 2′′-OH on the acceptor ADPR for PAR chain extension or branching, respectively (Figure 1A, B). Glu988 on PARP1 also facilitates the binding of NAD⁺ via an H-bond to the 3′-OH (Ruf et al., 1998). Consistent with Glu284 on HPF1, not Glu988 on PARP1, performing the de-protonation required for initiation, Ahel’s group (Fontana et al., 2017) observe HPF1-mediated incorporation of ADPR by the E988K mutant of PARP1, albeit at lower levels than wild-type.

Our most dramatic and unexpected finding is the extent by which HPF1 converts PARP1 from a polymerase that adds long polymers of ADPR onto itself or histones, into a hydrolase that generates free ADPR. Under our conditions, almost 90% of all the NAD⁺ consumed by PARP1 in the presence of nucleosome activators and HPF1 is converted to free ADPR (Figure 5). This observation holds true both at short time points of the reaction (Figure 5D) and at the end of the reaction where all the NAD⁺ has been consumed (Figure 6C). Also, recall that the presence of both nucleosomes and HPF1 increases the overall turnover of NAD⁺ by PARP1 (Figure 5C, Figure 6C). These dual effects lead to rapid depletion of NAD⁺ and formation of high concentrations of free ADPR. Given that increasing PARylation acceptor sites (i.e., high concentrations of H3 histone tail peptide, Figure 5D) greatly reduces the percentage of free ADPR formed, we conclude that HPF1 blocks PAR chain formation by occluding the extension site on PARP1 (Suskiewicz et al., 2020b), and thus PARP1, still bound to activating DNA (i.e., nucleosome), quickly runs out of suitable PARylation sites and therefore uses water as a nucleophile instead. The affinity of PARP1 for nucleosomes is very high (nM), and it is thought that significant PARylation is required for dissociation of DNA from PARP1 (Rudolph et al., 2021; Langelier et al., 2014). Thus, it is unclear what ratio of treadmilling would occur under cellular conditions where rapid exchange of PARP1 to other nucleosomes is most likely not occurring, and new methods will be needed to investigate this quantitatively. The high consumption of NAD⁺ by PARP1 suggests that PARP1 not only plays a role in the DNA damage response but also in metabolic homeostasis (Hurtado-Bagès et al., 2020; Murata et al., 2019). NAD⁺ is electron acceptor in glycolysis and the citric acid cycle. PARP1 is the major consumer of NAD⁺ in the nucleus, and the rapid depletion of the NAD⁺ pool (50–80%) by PARP1 in response to DNA damage leads to ATP depletion, has dramatic consequences for mitochondria, and leads to cell death via apoptosis and necrosis (Berger, 1985). The observation of partitioning away from PARylation and towards ADPR formation by HPF1 is a novel finding that suggests a more important role for ADPR than anticipated as a signaling molecule in its own right (Perraud et al., 2001; Qi et al., 2019).

The conversion of PARP1 into an NAD⁺ hydrolase is also evident in the much lower incorporation of chain ‘middles’ onto histones or PARP1 (Figure 6D), indicating that HPF1-mediated PARylation yields much shorter chains than the typical long chains seen during autoPARylation in the absence of HPF1. Our quantitative demonstration of these shorter chains is in agreement with previous observations that smears of PARP1 typically seen after autoPARylation in SDS-PAGE are reduced in the presence of HPF1 (Gibbs-Seymour et al., 2016) and that PAR chains are much reduced in amount and length as detected by enzymatic labeling of terminal ADPR (Ando et al., 2019). Additionally, it is interesting to compare our findings with a recent demonstration that HPF1-mediated PARylation in cells leads primarily to mono-ADPR on serine residues, in part due to the activity of the glycohydrolase PARG (Bonfiglio et al., 2020), which interestingly also generates ADPR. PARG has also been shown to mediate transPARylation of histones, in part by competing with PARP1 for PAR chains (Ménard et al., 1990; Thomassin et al., 1992; Lagueux et al., 1995). It therefore remains to be elucidated whether the rapid and sustained depletion of NAD⁺ after DNA damage in intact cells (Juarez-Salinas et al., 1979) is due to PARylation and subsequent dePARylation by PARG, PARylation, and futile treadmilling by PARP1 alone, or some combination of these events. As with the increased NAD⁺ hydrolase activity triggered by HPF1, the formation of shorter PAR chains is partially preserved using the catalytic HPF1 mutant E284A. As concluded for the high hydrolase activity, these shorter PAR chains using either wild-type HPF1 or its E284A mutation are readily understandable given that HPF1 partially occupies the binding site typically associated with PAR chain extension (Suskiewicz et al., 2020b). Although our assay did not allow us to measure chain branches with sufficient sensitivity, we would argue that for this same reason HPF1-mediated PARylation has few if any branch points.

The formation of shorter PAR chains in the presence of HPF1 has important consequences that become particularly relevant when considering intracellular concentrations of these proteins. PARP1 is a very abundant nuclear protein, exceeding the concentration of HPF1 by about twentyfold (Gibbs-Seymour et al., 2016). Also, PARP1 is one of the very first proteins recruited to DNA damage sites induced by laser microirradiation, whereas HPF1 arrives more slowly (Aleksandrov et al., 2018; Mahadevan et al., 2019). Thus, it is possible that substantial PARylation at some points in time, at some sites, or at some types of damage occurs without HPF1. The consequences for the so-called PAR code (Aberle et al., 2020; Karlberg et al., 2013) are immense. PARP1-alone-mediated autoPARylation leads to long and branched chains, whereas HPF1-mediated PARylation leads to short, most likely unbranched chains. This change in the PARylation pattern may result in the recruitment of different repair proteins to DNA damage sites with HPF1 vs. those without, and thus in turn may direct DNA damage to be repaired by one pathway over another. The differentiation between these two outcomes in a cellular context is difficult but worthy of further investigation.

All data generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided for all Figures.

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Author details

1. Johannes Rudolph

   Department of Biochemistry, University of Colorado Boulder, Boulder, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Methodology, Writing - original draft, Project administration, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-0230-3323

2. Genevieve Roberts

   Department of Biochemistry, University of Colorado Boulder, Boulder, United States

   Contribution

   Formal analysis, Investigation, Methodology

   Competing interests

   No competing interests declared

3. Uma M Muthurajan

   Department of Biochemistry, University of Colorado Boulder, Boulder, United States

   Contribution

   Investigation, Methodology, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-2596-5848

4. Karolin Luger

   Howard Hughes Medical Institute, University of Colorado Boulder, Boulder, United States

   Contribution

   Conceptualization, Resources, Supervision, Project administration, Writing - review and editing

   For correspondence

   karolin.luger@colorado.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-5136-5331

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