Quantitative site-specific ADP-ribosylation profiling of DNA-dependent PARPs

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Quantitative site-specific ADP-ribosylation profiling of DNA-dependent PARPs Jean-Philippe Gagné a , Chantal Ethier a , Daniel Defoy b , Sylvie Bourassa b , Marie-France Langelier c , Amanda A. Riccio c , John M. Pascal c , Kyung-Mee Moon d , Leonard J. Foster d , Zhibin Ning e , Daniel Figeys e , Arnaud Droit b , Guy G. Poirier a,∗ a

Centre de recherche du CHU de Québec – Pavillon CHUL, Faculté de Médecine, Université Laval, Québec, Canada Plateforme Protéomique du Centre de Recherche du CHU de Québec – Pavillon CHUL, Faculté de Médecine, Université Laval, Québec, Canada c Department of Biochemistry & Molecular Biology, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, USA d Department of Biochemistry and Molecular Biology, University of British Columbia, Centre for High-Throughput Biology, Vancouver, British Columbia, Canada e Ottawa Institute of Systems Biology, Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, Ontario, Canada b

a r t i c l e

i n f o

Article history: Received 16 January 2015 Accepted 4 February 2015 Available online xxx Keywords: PARP-1 PARP-2 PARP-3 Mass spectrometry Proteomics Poly(ADP-ribosyl)ation ADP-ribose Hydroxylamine Posttranslational modification

a b s t r a c t An important feature of poly(ADP-ribose) polymerases (PARPs) is their ability to readily undergo automodification upon activation. Although a growing number of substrates were found to be poly(ADPribosyl)ated, including histones and several DNA damage response factors, PARPs themselves are still considered as the main acceptors of poly(ADP-ribose). By monitoring spectral counts of specific hydroxamic acid signatures generated after the conversion of the ADP-ribose modification onto peptides by hydroxylamine hydrolysis, we undertook a thorough mass spectrometry mapping of the glutamate and aspartate ADP-ribosylation sites onto automodified PARP-1, PARP-2 and PARP-3. Thousands of hydroxamic acid-conjugated peptides were identified with high confidence and ranked based on their spectral count. This semi-quantitative approach allowed us to locate the preferentially targeted residues in DNA-dependent PARPs. In contrast to what has been reported in the literature, automodification of PARP-1 is not predominantly targeted towards its BRCT domain. Our results show that interdomain linker regions that connect the BRCT to the WGR module and the WGR to the PRD domain undergo prominent ADP-ribosylation during PARP-1 automodification. We also found that PARP-1 efficiently automodifies the D-loop structure within its own catalytic fold. Interestingly, additional major ADP-ribosylation sites were identified in functional domains of PARP-1, including all three zinc fingers. Similar to PARP-1, specific residues located within the catalytic sites of PARP-2 and PARP-3 are major targets of automodification following their DNA-dependent activation. Together our results suggest that poly(ADP-ribosyl)ation hot spots make a dominant contribution to the overall automodification process. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Poly(ADP-ribosyl)ation has long been recognized as a surveillance mechanism that contributes to preserve genomic stability [1]. Upon sensing DNA damage, poly(ADP-ribose) polymerases (PARPs) are activated and catalyze the formation of poly(ADPribose) polymers (pADPr) that act as a molecular scaffold promoting

∗ Corresponding author at: Centre de recherche du CHU de Québec – Pavillon CHUL, 2705 Boulevard Laurier, Québec, QC, Canada G1V 4G2. Tel.: +1 418 654 2267; fax: +1 418 654 2159. E-mail address: [email protected] (G.G. Poirier).

the accumulation of repair factors at DNA lesions [2]. Although considerable progress has been made in characterizing members of the PARP family, DNA-dependent PARPs (i.e. PARP-1, PARP-2 and PARP3) are still responsible for most of the pADPr synthesis in cells and remain the most important actors of the interplay between DNA damage response and pADPr-mediated pathways. The primary targets of the poly(ADP-ribosyl)ation reaction are the PARPs themselves through a phenomenon called automodification. This auto-catalytic activity is thought to proceed by either intramolecular (cis) or intermolecular (trans) mechanisms [3]. A variety of chromatin regulatory factors involved in the detection and signalling of DNA damage are also poly(ADP-ribosyl)ated on sites of DNA breaks by PARPs [4]. The addition of ADP-ribose

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polymers onto poly(ADP-ribosyl)ated substrates makes them particularly susceptible to three-dimensional structure alterations. The conformational changes induced by poly(ADP-ribosyl)ation are largely driven by the steric hindrance and the electrostatic perturbations induced by the local accumulation of ADP-ribose moieties. As a result, poly(ADP-ribosyl)ation is an important posttranslational modification that can significantly alter proteins ability to perform their functions. For example, automodification takes place at multiples sites within PARP-1, a phenomenon that explains the characteristic retardation pattern of extensively automodified PARPs in SDS-PAGE [5,6] and the dramatic decrease of PARP enzymatic activity [7]. Limited proteolysis analysis of poly(ADP-ribosyl)ated PARP-1 fragments showed that most of the automodification is located in a central region of the protein that overlaps with the BRCT domain [8–10]. However, a 40-kDa C-terminal fragment of PARP-1 was shown to be targeted by the catalytic activity of PARP-1, therefore challenging the concept of a unique BRCT-containing automodification domain [11,12]. This was further supported by another study in which the identification of both N-terminal DNA-binding domain and the C-terminal catalytic domains were demonstrated as targets for the automodification reaction [7]. As many as 28 poly(ADP-ribosyl)ation sites were estimated to reside into PARP1 by measuring its enzymological parameters [7] but this result likely underestimates the actual number of binding sites because of partial site occupancy. Although direct biochemical evidence was missing, PARP-1 automodification was postulated to occur on glutamate and aspartate residues on the basis of the esterlike bond established for the ADP-ribosylated core histones [13]. However, contradictory information add to the confusion regarding the nature of the pADPr linkage since it was reported that none of the glutamate residues within the central automodification domain were targeted by the poly(ADP-ribosyl)ation reaction. The authors rather suggested the resistance of automodified PARP-1 to hydroxylamine and that lysine side chains would be the major pADPr acceptors [14]. Hydroxylamine stability has been used to classify pADPr–protein bonds into sensitive and resistant linkages [15,16]. Carboxylester-type ADP-ribose–protein bonds formed with the side chain carboxylate group of glutamate and aspartate residues are the most susceptible to hydroxylamine hydrolysis. Toxin ADP-ribosyltransferase activity targeting the guanidino group of arginines and the thiol group of cysteines were also reported to be hydroxylamine-labile [17]. On the other hand, other types of linkages such as the ADP-ribose ketoamine conjugates (glycated lysine residues) are stable under the same conditions [18]. Current literature indicates that glutamate and aspartate residues are preferentially targeted by the ADP-ribose transferase activity of PARPs [19–23]. A proteome-wide analysis of the ADPribosylated proteome performed by Zhang and colleagues [20] based on the hydroxylamine sensitivity of carboxylester-type ADPribose–protein bonds formed following oxidative DNA damage and PARP activation revealed that hundreds of PARP substrates are covalently poly(ADP-ribosyl)ated. Consistent with earlier studies, PARP-1 automodification was reported to occur at several sites [21–23]. The widespread automodification pattern of PARP-1 is also consistent with the activation mechanism of PARP-1 where its domains collapse on the DNA break, a conformation that promote interdomains contacts and automodification [24,25]. Current efforts were focused on the identification of ADPribosylation sites and the determination of the extent of poly(ADP-ribosyl)ated residues within DNA-dependent PARPs by mass spectrometry. In vitro automodified PARP-1, PARP-2 and PARP-3 were treated with hydroxylamine to specifically convert the pADPr attached to glutamate and aspartate residues into a hydroxamic acid conjugate that generates a spectral signature

easily interpretable by common peptide annotation algorithms [20]. The rationale behind this approach was that the cumulative spectral count would serve as an index of ADP-ribosylation activity on targeted residues. High abundance poly(ADP-ribosyl)ated peptides were repeatedly identified from multiple iterations, providing a simple method to estimate preferred modification sites. Our results demonstrate that PARP-1 automodification is not restricted to an “automodification domain”, suggesting this definition might need to be changed. 2. Materials and methods 2.1. PARPs automodification and preparation for MS analysis Bovine PARP-1 was purified from calf thymus essentially as described by Zahradka and Ebisuzaki [26] up to the DNA–cellulose step (400 units/mg). Approximately 20 units of PARP-1 were incubated for 30 min at 30 ◦ C in a reaction buffer containing 100 mM Tris–HCl pH 8.0, 10 mM MgCl2 , 10% (v/v) glycerol, 10 mM DTT, 1 mM NAD, 10% (v/v) ethanol and 25 ␮g/mL calf thymus activated DNA (Sigma). Following the automodification reaction, during which PARP-1 catalyze the attachment of pADPr polymers onto itself, automodified PARP-1 was precipitated with isopropanol and sodium acetate as described by Brochu et al. [27]. The reaction mixture was kept on ice for 2 h and then centrifuged at 10,000 × g for 10 min at 4 ◦ C. The pellet was washed 2 times with ice-cold 80% (v/v) ethanol and resuspended in 100 ␮L of buffer containing 50 mM Tris–HCl pH 8.0 and 8 M urea. PARP-1 was reduced with the addition of 10 mM DTT for 20 min and alkylated with 15 mM iodoacetamide for 30 min. Urea concentration was lowered to 1 M with the addition of 7 volumes of 50 mM Tris–HCl pH 8.0. PARP-1 was then subjected to overnight proteolytic digestion by the addition of 1 ␮g of a trypsin/Lys-C mixture (Promega). ADP-ribosylated peptides were converted to hydroxamic acid-conjugated peptides following overnight incubation with 1 M hydroxylamine. Peptides were isolated on C18 spin columns according to the manufacturer’s instructions (Thermo Scientific) and dried to completion in a speed vac evaporator. Highly purified 6×-His and GST-tagged human PARP-1, PARP-2 and PARP-3 were also used to generate an ADP-ribosylation profile of DNA-dependent PARPs. Twenty micrograms of GST-tagged PARP-1, PARP-2, PARP-3 (BPS Biosciences) and 6×-His-tagged PARP-1, PARP-2 and PARP-3 purified as described in [28–30] were automodified in the automodification reaction buffer in presence of NAD and calf thymus activated DNA. To establish a correlation between the spectral count of hydroxamic acid signatures and the degree of PARP-1 automodification, highly purified recombinant human PARP-1 (Enzo Life Sciences) was pre-incubated with PARP-1 inhibitors AG14361 and BMN-673 (Selleckchem) for 30 min at 30 ◦ C in the reaction buffer devoid of NAD. Following the pre-incubation with the inhibitors, NAD was added to the reaction and samples were processed as described. 2.2. Evaluation of automodified PARP-1 sensitivity to hydroxylamine The susceptibility of PARP-1–ADP-ribose linkages towards hydroxylamine was monitored by slot-blot assays, SDS-PAGE protein staining and Western blot. Radiolabeled automodified bovine PARP-1 was synthesized as described previously with the addition of 75 ␮Ci of 32 P-labelled NAD (800 Ci/mmol, PerkinElmer) in the reaction buffer. 32 P-labelled automodified PARP-1 was either resuspended in 1 M hydroxylamine or 50 mM Tris–HCl pH 8.0 solutions for 5, 15, 30 60, 120 min or overnight. Aliquots were mixed with 200 ␮L TBS (20 mM Tris–HCl pH 7.5, 500 mM NaCl)

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and transferred onto nitrocellulose membrane using a Bio-Dot® SF manifold micro-filtration apparatus (Bio-Rad) equipped with a filtration template for slot-blots. Slots were washed twice with 500 ␮L TBS. The membrane was air-dried and PARP-1 bands were developed on a Typhoon 9200 PhosphorImager and quantified with ImageQuant software version 5.2 (Molecular Dynamics). To monitor the electrophoretic mobility of automodified PARP1 on SDS-PAGE, highly purified recombinant human PARP-1 (Enzo Life Sciences) was automodified in the reaction buffer described previously and subjected to hydroxylamine hydrolysis by incubating the reaction products for 5, 15, 30, 60, 120, 240 min or overnight with a final concentration of 1 M hydroxylamine. Samples were resolved on a 4–12% CriterionTM XT Bis–Tris gradient gel (Bio-Rad) and stained with Sypro Ruby (Bio-Rad) according to the manufacturer’s instructions. Images were acquired using a Geliance CCD-based bioimaging system (PerkinElmer). Corresponding samples were also analyzed by Western-blot using the mouse monoclonal antibody clone C2-10 against PARP-1 to validate that hydroxylamine hydrolysis of automodified PARP-1 restores PARP-1 to its original migration position. 2.3. Liquid chromatography–tandem mass spectrometry (LC–MS/MS) All spectral count-based quantitation of ADP-ribosylated residues were performed at Plateforme Protéomique du Centre de Recherche du CHU de Québec, Canada. Mass spectrometry analysis was performed on a TripleTOF® 5600 mass spectrometer fitted with a nanospray III ion source (ABSciex, Concord, ON) and coupled to an Agilent 1200 HPLC. Samples were injected by the Agilent 1200 autosampler onto a 0.075 mm (internal diameter) self-packed PicoFrit column (New Objective) packed with a isopropanol slurry of 5 ␮m Jupiter C18 (Phenomenex) stationary phase using a pressure vessel set at 700 p.s.i. The length of the column was 15 cm. Samples were run using a 65 min gradient from 5 to 35% solvent B (solvent A: 0.1% formic acid in water; solvent B: 0.1% formic acid in acetonitrile) at a flow rate of 300 nL/min. Data was acquired using an ion spray voltage of 2.4 kV, curtain gas of 30 PSI, nebulizer gas of 8 PSI and an interface heater temperature of 125 ◦ C. An information-dependant acquisition (IDA) method was set up with the MS survey range set between 400 and 1250 amu (250 ms) followed by dependent MS/MS scans with a mass range set between 100 and 1800 amu (50 ms) of the 20 most intense ions in the high sensitivity mode with 2+ to 5+ charge state. Dynamic exclusion was set for a period of 3 s and a tolerance of 100 ppm. MGF peak list files were created using Protein Pilot version 4.5 (ABSciex) utilizing the Paragon and Progroup algorithms [31]. An independent laboratory validation study was conducted for assessing the accuracy of hydroxamic acid modification assignments using high mass accuracy mass analyzers (Supplementary Materials & Methods). 2.3.1. Database searching Combined MS/MS datasets were analyzed using Mascot (Matrix Science, London, UK; version 2.4.1) and X! Tandem (The GPM, thegpm.org; version CYCLONE (2010.12.01.1)). Mascot was set up to search the Bos taurus UniREF100 database (2013-11–18, 28,128 entries) assuming the digestion enzyme trypsin. X! Tandem was set up to search a subset of the same database also assuming trypsin. Mascot and X! Tandem were searched with a fragment ion mass tolerance of 0.100 Da and a parent ion tolerance of 0.100 Da. Carbamidomethyl of cysteine was specified in Mascot and X! Tandem as a fixed modification. Methionine oxidation, pyroglu formation (from N-terminal Glu or Gln), hydroxamic acid-modification of aspartic acid and glutamic acid, ammonia loss of the N-terminus

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and acetyl modification of the protein N-terminus were specified as variable modifications. Tandem mass spectra of highly purified recombinant GST- and 6X-HIS-tagged PARPs were searched against each corresponding PARP protein sequence using the same database searching parameters. 2.3.2. Criteria for MS/MS spectrum annotation and spectral count Scaffold (version Scaffold 4.2.0, Proteome Software Inc., Portland, OR) was used to validate MS/MS based peptide and protein identifications. Peptide identifications were accepted if they could be established at greater than 95.0% probability by the Scaffold Local FDR algorithm [32]. Protein identifications were accepted if they could be established at greater than 95.0% probability and contained at least 2 identified peptides. Protein probabilities were assigned by the Protein Prophet algorithm [33]. Proteins that contained similar peptides and could not be differentiated based on MS/MS analysis alone were grouped to satisfy the principles of parsimony. Proteins sharing significant peptide evidence were grouped into clusters. Spectral counting of individual ADP-ribosylation sites was performed using data from Scaffold modelling. Peptides bearing a hydroxamic acid signature (+15.0109 Da) assigned by Scaffold were retained only if their corresponding Mascot ion score was ≥30. 3. Results 3.1. ADP-ribose–protein bonds onto PARP-1 are highly reactive to hydroxylamine Although abundantly documented in the literature, we sought to re-evaluate the sensitivity of in vitro poly(ADP-ribosyl)ated PARP-1 to hydroxylamine. As a first quantitative estimation of the hydroxylamine sensitivity of automodified PARP-1, we generated a 32 P-labelled automodified PARP-1 that we subjected to hydroxylaminolysis from few minutes to overnight (Fig. 1A). Reaction products were slot-blotted on a nitrocellulose membrane. In this assay, a mixture of pADPr and PARP-1 protein is passed through a nitrocellulose filter which result in the retention of the protein on the membrane while the pADPr will pass through. After washing and drying, the membrane is exposed to phosphor-imaging screens for quantitation. As expected, chains of pADPr are rapidly detached from automodified PARP-1 following incubation with 1 M hydroxylamine. Almost half of 32 P-labelled pADPr is detached from PARP-1 in the first 5 min of exposure to hydroxylamine. The labelling then slowly decreases down to approximately 15% after overnight exposure. This result indicates that most of ADP-ribose–protein bonds onto PARP-1 are linked through a hydroxylamine-sensitive bond. Accordingly, we estimate that approximately 85% of the linkages are ester-type ADP-ribose–protein bonds targeting glutamate and aspartate residues. The presence of residual hydroxylamineresistant bonds was also observed on histones, automodified PARP-1 and poly(ADP-ribosyl)ated nuclear proteins [13,15,34], suggesting that ADP-ribosylation can occur via alternative linkages such as ketoamine (lysine/arginine) or thioglycoside (cysteine) bonds. To further characterize the effect of hydroxylamine on automodified PARP-1, reaction products were also resolved by SDS-PAGE prior to immunoblot analyses using antibodies against PARP-1 (Fig. 1B). Following the addition of long chains of pADPr onto itself, PARP-1 undergoes a massive electrophoretic mobility shift and is no longer detected by the anti-PARP-1 monoclonal antibody C2-10, most likely because of epitope masking. Following the hydroxylaminolysis of automodified PARP-1, the detachment of pADPr chains from PARP-1 unmasks the epitope and a typical PARP-1 smear is observed. As treatment with hydroxylamine continues,

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Fig. 1. Automodification of PARP-1 mostly occurs via carboxylester-type ADP-ribose–protein bonds. (A) The release of hydroxylamine-sensitive linkages was monitored by slot-blot analysis. Radiolabeled automodified PARP-1 was incubated with hydroxylamine or a Tris-buffered solution and the reaction products were slot-blotted onto protein binding nitrocellulose membrane. Radiolabeled protein-free pADPr polymers are filtered through the membrane while PARP-1 protein is retained. The residual radioactivity, which correspond to hydroxylamine-resistant ADP-ribose–protein bonds, was measured by autoradiography on a phosphorimager and plotted as a function of time (right panel). (B) The loss of pADPr polymers onto automodified PARP-1 was monitored by electrophoretic mobility shift analysis. Automodified PARP-1 supershift is rapidly decreased following hydroxylamine hydrolysis as visualized by the reappearance of a 113-kDa band on SDS-PAGE stained with Sypro Ruby fluorescent stain. Corresponding extracts were analyzed by Western blot using the anti-PARP-1 monoclonal antibody clone C2-10.

the PARP-1 smear gradually resumes to a single 113-kDa protein band, an observation that indicates an efficient de-poly(ADPribosyl)ation of automodified PARP-1. Since hydroxylamine do not hydrolyze the pADPr but rather detaches it from the protein as the consequence of its reactivity to acyl groups, the total amount of pADPr in the samples is unchanged as visualized by western blot using the anti-pADPr polyclonal antibody clone 96-10 (Supplementary Fig. S1). Altogether, these results re-establish the hydroxylamine sensitivity of PARP-1 and the predominant formation of carboxylesters linkages of protein-ADP-ribose. 3.2. Spectral count of hydroxamic acid signatures correlates with the level of PARP-1 automodification Having established that the release of hydroxylamine-labile ADP-ribose linkages is sufficient to revert automodified PARP-1 to a single protein band corresponding to its unshifted form, we sought to evaluate the quantitative relationship between the level of PARP-1 automodification and the number of hydroxamic acid

signatures that can be identified by mass spectrometry. As one can expect during the course of PARP-1 automodification, the ratio of ADP-ribosylated residues is predicted to increase from low to high stoichiometry to reflect the overall automodification process. To validate this hypothesis, we produced different levels of automodified recombinant human PARP-1 as a consequence of partial PARP-1 inhibition by AG14361 (Fig. 2). The reaction of PARP-1 automodification in the presence of increasing amounts of PARP-1 inhibitor produces intermediary products as it can be visualized by the increase of PARP-1 mobility in gel. In this study, all ADP-ribosylated PARPs samples were processed with a combination of in-solution Lys-C/trypsin dual proteases digestion which provides a convenient and reproducible method for sample preparation and mass spectrometry. The number of hydroxamic acid signatures identified by mass spectrometry recapitulates the level of PARP-1 automodification (Fig. 2) therefore making it a valuable tool to estimate its degree of activation. Additionally, in-gel tryptic digestion was similarly efficient for the detection and identification of hydroxamic acidmodified peptides by mass spectrometry (Supplementary Fig. S2).

Fig. 2. Label-free spectral count analysis of hydroxamic acid-modified peptides is quantitatively correlated to the level of PARP-1 automodification. PARP-1 was automodified in presence of increasing concentrations of AG14361 to generate a range of automodification levels as visualized by SDS-PAGE and Sypro Ruby staining. Reaction products were in-solution digested with trypsin and Lys-C and subjected to hydroxylamine hydrolysis. Peptides were isolated on C18 microspin columns and analyzed by LC–MS/MS. The number of tandem mass spectra confidently assigned with a hydroxamic acid signature was plotted with respect to each reaction (right panel).

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3.3. Site-specific ADP-ribosylation on glutamate and aspartate residues is located throughout PARP-1 conserved domains with the exception of the BRCT domain Based on our label-free and count-based quantitative strategy, we undertook a large-scale proteomics survey to identify PARP-1 ADP-ribosylation sites. Our idea was to perform a comprehensive re-evaluation of the actual targeted residues into PARP-1. We used a semi-purified PARP-1 isolated by DNA-cellulose chromatography from calf thymus according to the method developed by Zahradka and Ebisuzaki [26]. This approach has both the advantages of providing good protein amounts required for multiple rounds of mass spectrometry analysis while preserving PARP-1 in a more physiological context. Endogenously expressed PARP-1 protein complexes are more likely to retain full activity compared to recombinant PARP-1 expressed in bacterial and eukaryotic systems which possess limitations that may explain its common lower activity. For example, studies have shown that histones can stimulate PARP-1 activity up to 20-fold [35]. Although alternative approaches were developed to identify ADP-ribosylated residues by mass spectrometry (reviewed in [21]), including the conversion of the pADPr to ribose-5 -phosphate that we described earlier [22], the generation of a hydroxamic acid signature specific to glutamate and aspartate developed by Zhang et al. [20] is more appropriate for a thorough analysis of PARP-1 automodification since: (i) a simplification of the ADP-ribose adduct based on a chemical conversion with hydroxylamine is straightforward compared to enzymatic reactions that require purified snake venom phosphodiesterase; (ii) major poly(ADP-ribosylation) sites are hydroxylamine-labile since it restores PARP-1 electrophoretic mobility to a comparable state as unmodified PARP-1 and (iii) the fragmentation of hydroxylamine-derivatized peptides yields easily interpretable peptide mass spectra that do not require any a priori information regarding the complex atypical fragmentation scheme of ADP-ribosylated or ribose-5 -phosphate-modified peptides. The small +15 Da increment of hydroxamic acid-conjugated residues do not interfere with peptide ionization of fragmentation behaviour, thus making it a valuable strategy to evaluate ratios of modified to unmodified PARP-1 tryptic peptides. Knowing that hydroxylamine does not react non-specifically with unmodified aspartate and glutamate residues [20], this method provides a robust, efficient and truly representative strategy to evaluate PARP-1 site-specific automodification. Stringent data filtering criteria based on the valid identification probabilities computed by PeptideProphet [32] and ProteinProphet [33] algorithms (implemented in Scaffold) were initially applied to achieve confident peptide identifications with low false discovery rate after LC–MS/MS analysis and database search by Mascot. Peptide and protein identity thresholds were conservatively set to a minimum of 95%. Using combined iterative analysis through multiple injections of in-solution digested automodified PARP-1, we first identified a total of 2053 hydroxamic acid signatures out of 14 999 PARP-1 tandem mass spectra (Supplementary Table S1). Then, all spectra that match to this set of confidently identified peptides were also required to exceed specific database search engine thresholds using additional acceptance criterion. Setting the threshold to a minimum Mascot ion score of ≥30 allowed 1251 ADP-ribosylation hits to be accepted. Poor quality spectra typically receive a lower Mascot ion score which reflects the number of matching fragment ions to a theoretical peptide MS/MS spectrum. These very strict parameters were selected to isolate high quality spectra with good signal-to-noise and to improve spectral count accuracy. Besides the identification of PARP-1 ADP-ribosylated peptides, we also assigned hundreds of hydroxamic acid signatures onto histones. ADP-ribosylation of the linker histone H1 and the core histones H2B were, by far, the

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most abundant ADP-ribosylated proteins co-purified with bovine PARP-1 (data not shown), a result absolutely consistent with previous studies which identified H1 and H2B as the best acceptors of pADPr in vitro [36] and in native chromatin [37] (reviewed in [38]). Corresponding bovine D/E ADP-ribosylation sites were distributed along human PARP-1 modular structure (Fig. 3). In order to maintain high stringency for listing accurate proteomic data, a residue is reported as ADP-ribosylated only if three tandem mass spectra pass the filtering criteria. Globally, 48 ADP-ribosylation sites were filtered out using a combination of these three cut-off scores (Table 1). Most of the ADP-ribosylation sites were identified in previous mass spectrometry-based studies thus validating the robustness of our approach [19–23]. However, our spectral count approach allows us to rank ADP-ribosylation sites rather than just providing a listing. In contrast to the current model, we found no significant ADPribosylation sites into PARP-1 BRCT domain. For decades, the BRCT and PARP-1 automodification domain (AD) were assumed to overlap [8]. Our results rather indicate that the primary region for PARP-1 automodification is located in the interdomain linker region that connects the BRCT and the WGR domains. One third of all hydroxamic acid signatures (457 spectra out of 1251) lie within a region encompassing residues 456–491. Together, residues E484, E488 and E491 forms the most notable PARP-1 automodification cluster (Table 1). Another ADP-ribosylation hotspot is localized in an additional hinge region that links the WGR and the PRD domains. This region contains amino acid E642 which is one of the most abundant residue targeted by the automodification activity of PARP-1 (Table 1). Interestingly, a major ADP-ribosylation site was unexpectedly identified in the catalytic domain of PARP-1 at amino acid E883. This position is located in the D-loop of PARP-1 which is an integral component of the catalytic fold [39]. It is also particularly interesting to note that all three PARP-1 zinc fingers are relatively good targets of its automodification activity, an observation consistent with the rapid dissociation of PARP-1 from DNA upon production of pADPr [28]. 3.4. PARP-1 ADP-ribosylation is highly resistant to inhibition Our spectral count-based approach used to generate an index of PARP-1 ADP-ribosylation sites led us to reason that preferentially targeted amino acids will be the most resistant to enzymatic inhibition since these sites are presumably positioned in a very favourable local environment. In a first set of experiments, we assessed the inhibitory potential of two highly potent PARP-1 inhibitors in our in vitro automodification assays with large amounts of purified 6X-HIS-PARP-1 required for further mass spectrometry analysis. As expected, both inhibitors (AG14361 and BMN-673) inhibit PARP-1 automodification although with different efficiencies (Fig. 4A, upper panel). As mentioned for Fig. 1B, automodified PARP-1 is not recognized by the anti-PARP-1 monoclonal antibody clone C2-10 so positive detection with this antibody only appears upon inhibition of PARP-1 at high concentrations (Fig. 4A, lower panel). More importantly, very little residual ADP-ribosylation persists upon PARP-1 inhibition with high concentrations of BMN-673 as visualized by 96-10 immunobloting (Fig. 4A, upper panel). The extensive decrease of PARP-1 automodification correlates with the concomitant reduction of hydroxamic acid signatures found in the presence of PARP inhibitor (Fig. 4B). Our attention was directed to examining the ADP-ribosylation of PARP-1 at automodification hotspots in the presence of high concentration of BMN-673. As expected, most ADP-ribosylation sites were sensitive to BMN-673 inhibition. Although PARP-1 automodification was massively abrogated in presence of 10 ␮M BMN-673 in

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Fig. 3. Quantitative profile of site-specific glutamate and aspartate ADP-ribosylation upon PARP-1 automodification. Affinity-purified bovine PARP-1 isolated from thymus gland tissue was automodified in vitro and analyzed by mass spectrometry. After trypsin and Lys-C protease digestion, peptides were incubated overnight with 1 M hydroxylamine to convert ADP-ribose linkages to hydroxamic acid conjugates. Peptides were isolated and cleaned on C18 microspins and analyzed by LC–MS/MS on a AB SCIEX 5600 TripleTOF with CID fragmentation. A spectral counting-based quantification methodology was used to evaluate the abundance of ADP-ribosylated sites. For convenience and comparison requirements, corresponding human PARP-1 amino acids positions were depicted. A schematic representation of human PARP-1 is illustrated with its conserved functional domains. ADP-ribosylated amino acid are positioned relative to PARP-1 protein sequence and the dark lines are proportional to the number of confidently assigned tandem mass spectra identified with a hydroxamic acid spectral signature (Spectral count). ZN: zinc finger; BRCT: BRCA1 C-terminus domain; WGR: conserved trytophan/glycine/arginine domain; PRD: PARP regulatory domain; CAT: catalytic domain. The white bars in the catalytic domain represent the conserved PARP activity signature sequence (H862-Y896-E988 triad). Domain boundaries were assigned according to Hassa P.O. [56]. Refer to Table 1 for a listing of site-specific ADP-ribosylated residues and Supplementary Table S1 for a complete spectral report.

Fig. 4. In vitro PARP automodification is resistant to high concentrations of inhibitors. (A) Comparative evaluation of in vitro PARP-1 inhibition with BMN-673 and AG14361. Highly purified human PARP-1 was automodified in presence of increasing concentration of inhibitors. The presence of pADPr polymers was revealed by Western blot using the anti-pADPr polyclonal antibody clone 96-10. The immunoreactivity of the anti-PARP-1 monoclonal antibody clone C2-10 was also assayed by Western blot on corresponding reaction products. (B) Analysis of residual PARP automodification in presence of BMN-673. The electrophoretic mobility shift of PARP-1 was visualized on SDS-PAGE stained with Sypro Ruby. The consequence of PARP-1 inhibition with BMN-673 on PARP-1 mobility was compared to AG14361 under the same conditions. The graph on the right panel shows spectral counts of peptides where ADP-ribosylated amino acids were detected for the automodification assays performed in presence of BMN-673. Refer to Supplementary Table S2 for a complete spectral report.

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J.-P. Gagné et al. / DNA Repair xxx (2015) xxx–xxx Table 1 Summary of ADP-ribosylation sites identified onto automodified calf thymus PARP1 based on assignments of hydroxamic acid signatures into tandem mass spectra. The number of individual MS/MS spectra is indicated for each position. AA

Positiona

95% minimum probabilityb

95% minimum probability + Mascot ion score >30c

Previous identification

E D E D D D E D E E D E D E D E E E E D E E D E D D E D D E E D E E D D E D D E E E D D E E E E D D E E D E D D E E D E E D D E E E E E D E D

3 6 26 31 45 68 70 72 76 90 100 107 112 147 155 168 169 175 190 191 193 205 211 212 214 217 218 243 260 263 276 281 296 297 307 310 360 387 406 407 448 456 457 461 471 484 488 491 534 536 540 547 561 576 577 578 580 608 609 612 642 644 648 649 650 680 688 690 692 715 726

79 2 3 1 49 15 5 1 37 27 8 4 1 72 3 6 36 6 63 15 5 7 5 24 2 4 5 5 5 12 58 77 36 9 5 8 70 8 1 1 3 78 28 83 97 116 105 164 1 3 35 4 13 1 7 1 32 8 1 2 172 27 25 14 27 3 14 30 1 11 2

66 0 0 1 21 3 3 0 30 27 7 3 0 35 0 4 18 2 50 8 1 4 4 18 0 2 2 0 1 6 50 45 19 2 3 1 4 1 1 0 3 69 16 64 58 77 77 96 0 0 20 0 3 1 5 1 28 3 0 1 123 6 11 7 8 2 5 9 0 0 1

[21]

[19,20] [20]

[19,21,22] [20] [19–21] [21] [20] [19–22] [19,20]

[20] [20] [20]

[20] [20] [20] [20,23]

[20] [20,22] [20] [20,22] [19–21] [19–21] [19–23] [19–23]

[20] [20] [20] [20] [20,22]

[19,20] [20] [19,20] [19] [20]

7

Table 1 (Continued) AA

Positiona

95% minimum probabilityb

D E D D D E D D E E D E D E E D E

731 772 783 784 788 795 805 807 809 812 830 832 835 840 883 899 923

1 1 5 5 3 2 1 1 30 7 7 12 29 2 121 6 1

95% minimum probability + Mascot ion score >30c 0 1 4 3 0 1 0 1 26 2 1 8 14 1 83 0 1

Previous identification

[22] [22]

[22]

a

Positions indicated refer to human PARP-1. Cut-off of 95% probability based on Scaffold statistical modelling (Peptide Prophet). c Additional cut-off criteria for tandem mass spectra and spectral count analysis. b

our in vitro assays, hydroxamic acid signatures can still be identified using stringent spectrum cut-off criteria (Supplementary Table S2). Hydroxamic acid signatures can still be detected in the 35 amino acids-long connecting loop between the BRCT and WGR domains. Notably, ADP-ribosylation on E488 and E491 is preserved. Endogenous ADP-ribosylation at E488/E491 has never been observed on basal unstimulated PARP-1 so these sites represent specific markers of PARP-1 automodification in our conditions. Following the same idea, E883, a major ADP-ribosylation hotspot in the catalytic domain of PARP-1 can still be detected. However, ADP-ribosylation at E642 was not detected upon PARP-1 inhibition with BMN-673. Additional replicates would be required to truly reflect the reaction dynamics at specific D/E locations but these results indicate that residual PARP-1 activity can be present even in the absence of common PARP-1 automodification readouts (i.e. shift in PARP-1 electrophoretic mobility or immunological detection of pADPr). 3.5. Automodification of PARP-2 and PARP-3 show a preference for C-terminal domains Our high-throughput MS/MS analysis of bovine PARP-1 automodification has been a successful strategy to achieve extensive sequence coverage and to generate a large collection of tandem mass spectra with unique hydroxamic acid signatures. The isolation of PARP-1 from complex modules of bovine protein networks allowed the preservation of an enzyme with high specific activity. However, such an approach was not readily amenable to PARP-2 and PARP-3 studies. Instead, we relied on highly purified recombinant PARP-2 and PARP-3 for further analysis of PARP automodification by mass spectrometry. We first analyzed recombinant human PARP-1 to evaluate its automodification profile in light of the quantitative data acquired with thousands of bovine PARP1 tandem mass spectra. Actually, this validation approach was used to see if our spectral count methodology was still acceptable for the evaluation of quantitative site-specific ADP-ribosylation of PARPs in a context of limited hydroxamic acid-modified peptides. As shown in Fig. 5, the typical automodification profile of recombinant human PARP-1 can be observed even if the number of MS/MS spectra annotated with hydroxamic acid signatures is much lower to the one obtained with several iterations of bovine PARP-1 (Fig. 3). Notably, E488, E491, E642 and E883 have been identified as prominent ADP-ribosylation sites, a result highly consistent

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Fig. 5. Schematic spectral-count based ADP-ribosylation profiles of highly purified recombinant human PARP-1, PARP-2 and PARP-3 after DNA-dependent activation. PARPs were automodified in vitro in presence of NAD+ and calf thymus activated DNA and subjected to trypsin and Lys-C digestion. ADP-ribosylated peptides were converted to hydroxamic acid-modified peptides by overnight incubation with 1 M hydroxylamine, isolated on C18 resin and analyzed by LC–MS/MS on a AB SCIEX 5600 TripleTOF with CID fragmentation. A spectral counting-based quantification methodology was used to evaluate the abundance of ADP-ribosylated sites. ADP-ribosylated amino acid are positioned relative to each corresponding PARP amino acids sequence and the dark lines are proportional to the number of confidently assigned tandem mass spectra identified with a hydroxamic acid spectral signature (Spectral count). ZN, zinc finger; BRCT, BRCA1 C-terminus domain; WGR, conserved trytophan/glycine/arginine domain; PRD, PARP regulatory domain; CAT, catalytic domain. The white bars in the catalytic domain represent the conserved PARP activity signature sequence (PARP-1: H862Y896-E988; PARP-2: H428-Y462-E558 and PARP-3: H384-Y414-E514 triads). Domain boundaries were assigned according to Hassa P.O.[56]. Refer to Table-2 for a listing of site-specific ADP-ribosylated residues and Supplementary Table S3 for a complete spectral report.

with their previous identification as ADP-ribosylation hotspots in bovine PARP-1 (Table 2). Thus, although the spectral coverage is much more limited, these experimental spectral count-based values were sufficient to recognize major ADP-ribosylation regions of PARP-1. The analysis of PARP-2 and PARP-3 automodification was therefore based on the same assumption. Even though we did not achieved in-depth ADP-ribosylation analysis as for PARP-1, the same stringent MS/MS cut-off criteria were applied to PARP-2 and PARP-3 proteomic datasets (Tables 3 and 4). In contrast to PARP-1 where several ADP-ribosylation sites are found in its N-terminal region, preferential ADP-ribosylation of PARP-2 and PARP-3 seems to occur in their C-terminal regions (Fig. 5). Interestingly, residue E449 of PARP-2, which corresponds to the evolutionarily conserved and highly ADP-ribosylated D-loop residue E883 of PARP-1, is also one of the most targeted amino acid. PARP-3 is also ADPribosylated in its catalytic domain, especially at residues E344 and D467. Again, several PARP-3 ADP-ribosylation sites reported in this

study were recently identified [40], thus providing inter-laboratory validation. 3.6. The catalytic domains of DNA-dependent PARPs are ADP-ribosylated One of the most striking observation in this study is the consistent identification of major ADP-ribosylation sites in the catalytic domains of PARP-1 (E883), PARP-2 (E449) and PARP3 (E344 and D467). Earlier we identified E883 as a covalently ADP-ribosylated residue by targeting the ribose-5 -phosphate remnant tag generated after the disruption of the phosphodiester bond of poly(ADP-ribosylated) residues [22]. However, this site has never been reported by other groups. Among those sites, only residue E344 of PARP-3 has been reported to reside in the catalytic domain of a DNA-dependent PARP [40]. We therefore aimed to evaluate if this unique observation could be attributable

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J.-P. Gagné et al. / DNA Repair xxx (2015) xxx–xxx Table 2 Summary of ADP-ribosylation sites identified onto highly purified recombinant human PARP-1 based on assignments of hydroxamic acid signatures into tandem mass spectra upon automodification. The number of individual MS/MS spectra is indicated for each position. PARP-1

9

Table 3 Summary of ADP-ribosylation sites identified onto highly purified recombinant human PARP-2 based on assignments of hydroxamic acid signatures into tandem mass spectra upon automodification. The number of individual MS/MS spectra is indicated for each position. PARP-2

AA

Position

95% minimum probabilitya

95% minimum probability + Mascot ion score >30b

Previous identification

AA

Position

95% minimum probabilitya

95% minimum probability + Mascot ion score >30b

E E D D E D E D E D E E E E D E D E E D E E D D E D D E E E E D E E D E E E D E E D D E E E E D E E D D E E E D E D D

3 26 45 72 76 112 116 145 147 155 168 169 175 190 191 212 217 218 276 285 296 360 387 406 456 457 461 471 484 488 491 536 540 576 577 578 580 608 609 612 642 644 648 649 650 680 715 756 763 795 805 807 809 810 812 835 883 899 914

12 1 1 1 11 5 4 3 49 12 2 21 1 55 8 3 1 10 2 3 4 3 1 2 18 6 6 10 1 29 35 4 8 1 3 1 14 1 3 1 31 4 5 4 5 2 5 2 2 5 1 6 13 1 4 2 26 9 1

12 0 0 1 9 5 1 3 36 1 0 2 0 32 3 1 0 6 2 0 1 1 0 0 16 4 5 5 1 22 30 1 6 0 3 0 9 0 0 1 24 2 2 3 2 1 3 0 1 3 0 1 2 0 0 1 18 1 0

[21]

E E D D E E E E E D E E E E E E D E D E E D E

97 138 207 214 215 216 221 222 231 235 252 300 304 341 350 355 359 378 396 418 449 465 489

1 2 5 2 2 1 1 19 15 9 8 1 3 2 22 9 20 5 6 1 33 17 2

0 2 0 0 1 1 1 7 13 1 5 1 1 2 17 4 10 0 3 0 27 8 1

[19,20]

[19,21,22] [20] [19–21] [21] [20] [19–22] [19,20] [20] [20] [20] [20]

[20] [20,23] [20,22] [20] [20,22] [19–21] [19–21] [19–23] [19–23] [20] [20] [20] [20]

[19,20] [20] [19,20] [19] [20]

[22] [22]

[22]

a Cut-off of 95% probability based on Scaffold statistical modelling (Peptide Prophet). b Additional cut-off criteria for tandem mass spectra and spectral count analysis.

Previous identification

a Cut-off of 95% probability based on Scaffold statistical modelling (Peptide Prophet). b Additional cut-off criteria for tandem mass spectra and spectral count analysis.

to an instrument-dependent bias even though the rest of our ADP-ribosylation dataset demonstrates consistency and robustness towards currently known modification sites. We selectively used high mass accuracy instruments located in multiple independent laboratories and using different peptide assignment algorithms to validate this observation. As expected, we were able to confidently assign hydroxamic acid modifications on E883 using all these instruments: a Thermo Scientific Orbitrap VelosTM , two Thermo Scientific Q-ExactiveTM located in two independent laboratories and a Thermo Scientific Orbitrap FusionTM TribridTM system (Supplementary Table S4). Besides, we validated most of the other ADP-ribosylation sites using these high resolution mass analyzers with high mass accuracy. The ADP-ribosylated residue E883 is located in a particular environment within the tryptic peptide (879IAPPE*APVTGYMFGK-893). The glutamate residue is surrounded by prolines in both its N- and C-terminal sides. As has previously been extensively documented, fragmentation of internal prolines in peptides typically results in the generation of intense y-ions with a strong bias towards fragmentation on the N-terminal side [41]. As a consequence, cleavage of the P–E bond is very much reduced, as well as the P–V bond on the C-terminal side of proline (Supplementary Fig. S3). In this context, there will only be a few low-intensity fragment ions left over to confidently localize the ADP-ribosylated glutamate. Instead, the intense N-terminal proline fragmentation pattern generates a serie of alternative internal ions (PE*, PE*A, PPE*, PPE*A, PPE*APV, etc.) with m/z masses that perfectly match with a +15.0109 Da hydroxamic acid adduct (Supplementary Fig. S3). However, these atypical ions are not taken into account by MASCOT and lower scores are obtained since these relatively numerous and intense ions represent unmatched internal ions rather than typical b- or y-ion series. We believe that the absence of b- or y-ions matched to the ADP-ribosylated residue by site localization algorithms used in previous studies might have

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Table 4 Summary of ADP-ribosylation sites identified onto highly purified recombinant human PARP-3 based on assignments of hydroxamic acid signatures into tandem mass spectra upon automodification. The number of individual MS/MS spectra is indicated for each position. PARP-3 AA

Position

95% minimum probabilitya

95% minimum probability + Mascot ion score >30b

Previous identification

E E E D E D E E E D E E D D E E E E E E E D D E E E E D E D D D E

12 15 26 27 64 65 76 154 158 159 160 163 210 212 234 237 293 303 305 309 310 316 330 335 344 366 367 368 418 455 467 488 530

9 2 3 5 4 2 1 49 19 3 1 2 8 5 2 1 2 3 1 28 16 5 2 1 35 2 2 1 2 2 46 1 1

4 2 0 1 4 1 1 17 7 1 0 2 5 3 2 1 1 2 1 19 8 4 2 0 27 2 1 1 1 1 37 1 0

[40] [40] [40]

[40] [40]

[40] [40]

[40]

a Cut-off of 95% probability based on Scaffold statistical modelling (Peptide Prophet). b Additional cut-off criteria for tandem mass spectra and spectral count analysis.

hampered its confident identification. These circumstances are similar for the ADP-ribosylation site found in the catalytic domain of PARP-2 since the corresponding peptide is very similar to PARP1 (445-IAPPE*APITGYMFGK-459). In PARP-3, two sites within the CAT domain stand out as most frequently ADP-ribosylated (E344 and D467). As mentioned previously, the ADP-ribosylation at E344 (338-VIQTYLE*QTGSNH-350) has been identified by Vyas S. and colleagues [40]. In contrast to E344, D467 is located in a proline-rich environment (461-SPPPGFD*SVIAR-472) particularly prone to internal fragmentation. 4. Discussion Although of prime importance in the understanding of PARP1 activity regulation at the molecular level, the exact boundaries of a so-called “automodification domain” and the nature of the internal poly(ADP-ribosylated) residues have remained largely unaddressed. In this study, strong experimental evidence supporting hydroxylamine-sensitivity of automodified PARP-1 was first re-established. An extensive change in PARP-1 molecular mass that alters its electrophoretic mobility has always been a hallmark of automodification as a consequence of the covalent attachment of large polymers of ADP-ribose on itself. The extreme lability of these large polymers to hydroxylamine hydrolysis is chemically consistent with an ester-like linkage. Indeed, hydroxylamineinduced cleavage of the pADPr bonds rapidly restores normal

PARP-1 electrophoretic mobility. Our analysis does not rule out the possibility that other types of ADP-ribose linkages exist upon PARP-1 automodification. However, our results indicate that hydroxylamine-resistant ADP-ribose linkages do not significantly contribute to PARP-1 automodification. Indeed, if hydroxylamineresistant ADP-ribose linkages would have been associated with the attachment of large polymers, exposition to hydroxylamine alone would not have been sufficient to completely restore PARP-1 to its normal electrophoretic mobility. Also, our slot-blot assay, which estimates the residual incorporation of ADP-ribose after hydroxylamine hydrolysis, is very similar to what has been observed for ADP-ribosylated histone H2B [13] and poly(ADP-ribosyl)ated PARP-1 [34]. An almost complete de-poly(ADP-ribosylation) was also observed for automodified PARP-1 following incubation with hydroxylamine in gel-based [42] and TCA precipitation/filtration assays [43]. Mono- or oligo-ADP-ribosylation on residues other than aspartate and glutamate could however be functionally important for the regulation of PARP activity or the orchestration of the DNA damage response at DNA damage sites. Regulatory cross-talk between lysine ADP-ribosylation and other posttranslational modifications has notably been suggested for PARPs [14,44] and histones [45,46]. However, since the side chain carboxylate group of glutamate and aspartate are the best targets of the ADP-ribosylation reaction, how PARP-1 can catalyze multiple reactions within the same active site as part of its normal function remains an open question. One possibility would be the non-enzymatic generation of ketoamine glycation conjugates on lysine and arginine side chains. Activated PARP cleaves NAD+ into nicotinamide and ADP-ribose at a very high rate and the accumulation of ADP-ribose has been shown to be a potent histone glycation agent in vitro [47,48]. Given that glutamate and asparate ADP-ribosylation prevails under most PARP automodification conditions, we sought to determine whether a spectral count-based mass spectrometry method could be used for a quantitative assessment of PARP-1 automodification. Taking advantage of the method developed by Zhang and colleagues [20], we showed that the number of tandem mass spectra bearing an hydroxamic acid signature parallels the level of PARP-1 automodification. The results presented here represent the first detailed quantitative mapping of the ADP-ribosylation sites in full-length PARP-1. In contrast to the current literature, in which PARP-1 automodification is generally considered to be clustered in a defined “automodification domain”, our results indicate that all conserved domains of PARP-1, with the exception of the BRCT domain, can be targeted by its ADP-ribose transferase activity. At this point, we can only speculate whether PARP-1 ADP-ribosylation results from intramolecular automodification [25] or intermolecular interplay via its self-association [49,50] but these results challenge the concept of automodification as a phenomenon that would disrupt BRCT-mediated PARP-1 dimerization [51]. Our quantitative ADP-ribosylation mapping strategy showed that PARP-1 automodification resides predominantly in flexible loop regions between structured regions or domains. The most abundant ADPribosylated sites (E484/E488/E491) were identified in the hinge region (aa 456–491) that connects the BRCT to the WGR domain. Interestingly, this cis-ADP-ribosylation hotspot is localized in an unstructured region that is thought to be displaced and reoriented towards the catalytic site upon PARP activation [25,52]. The stoichiometrically favoured ADP-ribosylation found at residues E488/E491 is consistent with the fact that only these sites were found to be ADP-ribosylated in all mass spectrometry-based studies of PARP-1 automodification, whatever the strategy used to target this posttranslational modification [19–23]. Moreover, these ADP-ribosylated residues were found to be extremely sensitive to olaparib inhibition as we would expect for particularly favoured sites [20].

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Interestingly, the complete deletion of this region (aa 374–525) was shown to reduce ADP-ribose incorporation into PARP-1 by approximately 30% [23], an evaluation consistent with our spectral count approach which assigned it one third of all hydroxamic acid signatures. Additionally, our analysis indicates that E642 and E883 are consistently ADP-ribosylated to a greater extent upon PARP-1 activation. A common feature of the hyper-ADP-ribosylated hotspots is their localization in hinges that extrude from the PARP-1 body. ADP-ribosylation of E642 occurs in the inter-domain linker that joins the WRG to the helical subdomain of the C-terminal catalytic domain. Since the loop formed by amino acids 626–645 has been shown to play a critical role in the DNA-dependent activation of the poly(ADP-ribosyl)ation activity of PARP-1 [53], a post-translational modification in this region is likely to alter its automodification activity and underscore the importance of interdomain communication to PARP-1 activity [24,54]. As for E883, it is located in an extruding loop located within PARP-1 catalytic domain (D-loop) [39]. The D-loop is a rigid proline-rich region highly conserved between PARP-1 and PARP-2 but this area present diversity among PARPs [55]. It has been suggested that the high sequence variability of this region could serve as the basis for the development of selective PARP inhibitors [39]. It seems unlikely that E883 could reach the active site of the same PARP-1 molecule so perhaps this is an in trans ADP-ribosylation site. Collectively, the most common PARP-1 ADP-ribosylation sites identified in this study (E484/E488/E491/E642/E883) are all resident in solvent-exposed proline-containing loop structures, a characteristic consistent with the preferred proline-directed ADPribosylation sequence motif identified by Zhang and colleagues [20]. Finally, similar to PARP-1, PARP-2 and PARP-3 ADPribosylation sites are common in interdomain solvent-exposed flexible loops, i.e. the PRD-CAT linker region of PARP-2 and the WGR-PRD and PRD-CAT of PARP-3. Clearly, ADP-ribosylation sites analysis indicates that targeted amino acids are not randomly spread along the protein length but rather tend to be clustered within confined regions. Numerous ADP-ribosylation sites were identified in all three zinc finger domains of PARP-1 which can be viewed as a molecular switch that uncouples the relay of DNA-binding activation signals to the catalytic site. However, such N-terminal structures are absent in PARP-2 and PARP-3 so their automodification activity was expected to occur in the catalytic domain which covers the greatest part of both proteins. In contrast to PARP-1, the N-terminal regions of PARP-2 and PARP-3 are not strictly required for DNA-dependent activation [28]. They are rather primarily regulated through their WGR domain by an allosteric activation mechanism similar to PARP-1 that involves the destabilization of the PRD [28]. Favoured automodification sites are located in the C-terminus catalytic domains of PARP-2 (E449) and PARP-3 (E344/D467) which could represent another mechanism of regulating enzyme activity. Further supporting this notion was the identification of a major ADP-ribosylation site in the catalytic domain of PARP-1 (E883), which is consistent with earlier studies that reported C-terminal automodification of PARP-1 at physiological NAD+ concentrations [7,11,12]. Following hypermodification of PARP-1, the alternate NADase reaction becomes a major component of the enzyme activity [7]. Indeed, when fully automodified, an abortive enzyme-catalyzed reaction is observed as PARP-1 continues to hydrolyze NAD while polymer synthesis plateaus. In this condition, polymer elongation is blocked but concomitant enzymatic hydrolysis of the nicotinamide–ribose bond persists. We speculate that site-specific ADP-ribosylation in the catalytic fold of PARP-1 could be involved in the activity switch to abortive NAD hydrolysis. Alternate NADase activity, which locally increases the concentration of unbound

11

ADP-ribose when virtually all D/E substrates are modified, might also explain the presence of non-enzymatic glycation on lysine and arginine side chains. There needs to be further exploration to determine whatever the role of automodification is in the catalytic domains of DNA-dependent PARPs. In summary, our results have demonstrated the robustness and reproducibility of a simple spectral count-based approach for estimating quantitative distribution of ADP-ribosylation. The accuracy of hydroxamic acid-modified peptide assignments to tandem mass spectra was corroborated by the use of high-resolution mass spectrometry. More importantly, our data indicate that the profile of the fragment ion series that originates from a peptide bearing a hydroxamic acid signature can efficiently be generated with a common ion source and mass analyzer. A particular situation exists when a proline-rich hydroxamic acid-conjugated peptide ion undergoes unusual fragmentation. However, we have shown that, even if unpredicted high-intensity fragment ions tend to score poorly by Mascot, we were able to make confident assignments by observing a serie of internal fragments highlighted by Scaffold’s graphical representation of the MS/MS spectra. We believe that this simple method of spectral counting in conjunction with hydroxamic acid derivatization will be a fast and reliable method to quantitatively assess the ADP-ribosylation status of PARPs substrates. Conflict of interest The authors declare that they have no conflict of interest. Acknowledgements This work was supported by the Canadian Institutes of Health Research [grant number MO-178013 to GGP and MOP-77688 to LJF]. GGP holds a Canada Research Chair in Proteomics. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.dnarep. 2015.02.004. References [1] M. De Vos, V. Schreiber, F. Dantzer, The diverse roles and clinical relevance of PARPs in DNA damage repair: current state of the art, Biochem. Pharmacol. 84 (2012) 137–146. [2] J.P. Gagne, E. Pic, M. Isabelle, J. Krietsch, C. Ethier, E. Paquet, I. Kelly, M. Boutin, K.M. Moon, L.J. Foster, G.G. Poirier, Quantitative proteomics profiling of the poly(ADP-ribose)-related response to genotoxic stress, Nucleic Acids Res. 40 (2012) 7788–7805. [3] A. Burkle, L. Virag, Poly(ADP-ribose): PARadigms and PARadoxes, Mol. Aspects Med. 34 (2013) 1046–1065. [4] F.G. Sousa, R. Matuo, D.G. Soares, A.E. Escargueil, J.A. Henriques, A.K. Larsen, J. Saffi, PARPs and the DNA damage response, Carcinogenesis 33 (2012) 1433–1440. [5] K. Ueda, M. Kawaichi, O. Hayaishi, Poly(ADP-ribose) synthetase, in: O. Hayaishi, K. Ueda (Eds.), ADP-Ribosylation Reactions, Academic Press, London, 1982, pp. 117–155. [6] K. Yoshihara, T. Hashida, H. Yoshihara, Y. Tanaka, H. Ohgushi, Enzyme-bound early product of purified poly(ADP-ribose) polymerase, Biochem. Biophys. Res. Commun. 78 (1977) 1281–1288. [7] Y. Desmarais, L. Menard, J. Lagueux, G.G. Poirier, Enzymological properties of poly(ADP-ribose)polymerase: characterization of automodification sites and NADase activity, Biochim. Biophys. Acta 1078 (1991) 179–186. [8] M. Nishikimi, K. Ogasawara, I. Kameshita, T. Taniguchi, Y. Shizuta, Poly(ADP-ribose) synthetase. The DNA binding domain and the automodification domain, J. Biol. Chem. 257 (1982) 6102–6105. [9] I. Kameshita, Z. Matsuda, T. Taniguchi, Y. Shizuta, Poly (ADP-ribose) synthetase. Separation and identification of three proteolytic fragments as the substrate-binding domain, the DNA-binding domain, and the automodification domain, J. Biol. Chem. 259 (1984) 4770–4776. [10] H. Ushiro, Y. Yokoyama, Y. Shizuta, Purification and characterization of poly (ADP-ribose) synthetase from human placenta, J. Biol. Chem. 262 (1987) 2352–2357.

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Please cite this article in press as: J.-P. Gagné, et al., Quantitative site-specific ADP-ribosylation profiling of DNA-dependent PARPs, DNA Repair (2015), http://dx.doi.org/10.1016/j.dnarep.2015.02.004