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International Journal of Molecular Sciences logoLink to International Journal of Molecular Sciences
. 2023 May 20;24(10):9042. doi: 10.3390/ijms24109042

PARP3 Affects Nucleosome Compaction Regulation

Alexander Ukraintsev 1,, Mikhail Kutuzov 1,, Ekaterina Belousova 1, Marie Joyeau 1, Victor Golyshev 1, Alexander Lomzov 1, Olga Lavrik 1,*
Editors: Daniela Montesarchio1, Giorgio Dieci1
PMCID: PMC10219313  PMID: 37240388

Abstract

Genome compaction is one of the important subject areas for understanding the mechanisms regulating genes’ expression and DNA replication and repair. The basic unit of DNA compaction in the eukaryotic cell is the nucleosome. The main chromatin proteins responsible for DNA compaction have already been identified, but the regulation of chromatin architecture is still extensively studied. Several authors have shown an interaction of ARTD proteins with nucleosomes and proposed that there are changes in the nucleosomes’ structure as a result. In the ARTD family, only PARP1, PARP2, and PARP3 participate in the DNA damage response. Damaged DNA stimulates activation of these PARPs, which use NAD+ as a substrate. DNA repair and chromatin compaction need precise regulation with close coordination between them. In this work, we studied the interactions of these three PARPs with nucleosomes by atomic force microscopy, which is a powerful method allowing for direct measurements of geometric characteristics of single molecules. Using this method, we evaluated perturbations in the structure of single nucleosomes after the binding of a PARP. We demonstrated here that PARP3 significantly alters the geometry of nucleosomes, possibly indicating a new function of PARP3 in chromatin compaction regulation.

Keywords: nucleosome, atomic force microscopy, poly(ADP-ribose)polymerase, chromatin structure, DNA compaction

1. Introduction

DNA in eukaryotes is mostly packed into chromatin [1]. The compaction is implemented by chromatin proteins and is presumably regulated by modifications of nitrogenous bases in DNA or chromatin proteins. The compaction level can influence the expression of the affected genes via transcription regulation [2]. The basic unit of DNA compaction is the nucleosome. The main functions of nucleosomes are compaction and the protection of DNA and regulation of gene expression [3]. A nucleosome consists of 147 nt of DNA wrapped around a histone octamer consisting of two molecules of each of the following histones: H2A, H2B, H3, and H4. This nucleoprotein complex is also termed the nucleosome core particle (NCP). The structural and functional details are reviewed in Reference [3].

The higher compaction level is usually mediated by linker histone H1. This histone binds to an NCP in the entry–exit region, thus forming a chromatosome, and chromatosomes can then condense into fibers. This compaction of NCPs requires a certain density of DNA wrapping [4]. Parameters of the fiber may depend on the NCP compaction degree. For example, the replacement of the H3 histone with CenpA leads to less compacted NCPs [5,6]. This alteration probably results in an alternative type of NCP compaction in fibers [5]. Functions of different types of DNA compaction in chromatin are being debated. One of the known chromatin changes occurs in response to the binding of poly(ADP-ribose)polymerase 1 (PARP1) [7].

The diphtheria toxin-like ADP-ribosyltransferase (ARTD) family of proteins consists of 17 members. These proteins share the active site of the catalytic domain [8]. Three proteins from this family—PARP1, PARP2, and PARP3—are known to be DNA-damage-dependent. These PARPs activate in response to DNA damage. They catalyze the transfer of ADP-ribose from NAD+ to an acceptor molecule. Various proteins and DNAs can act as an acceptor for these PARPs [9,10,11]. PARP1 and PARP2 can synthesize long branched polymers of ADP-ribose (PAR), whereas PARP3 performs only mono(ADP-ribosyl)ation [12]. PARP1 and PARP2 are regulator proteins in base excision repair and double-strand break repair [13,14,15,16,17]. ADP-ribosylation can perform the function of an intracellular signal for the recruitment of DNA repair proteins. On the other hand, as a type of post-translational modification, ADP-ribosylation influences the properties of a target protein [18].

Several authors have revealed an interaction of the PARP1 protein with NCPs and have proposed that there is a change in the NCP structure as a result [19]. PARP1 can affect the chromatin structure via poly(ADP-ribosyl)ation (PARylation) [20]. Under PARylation conditions, PARP1 modifies histone H1, thus causing its dissociation. Recently, a protein involved in histone PARylation (HPF1) was discovered [21,22]. In the presence of this protein, PARP1 and PARP2 can modify (ADP-ribosyl)ate core histones in an NCP. These modifications lead to chromatin relaxation [23].

PARP1 can also directly affect NCPs. It has been shown that in the absence of linker regions, PARP1′s binding to an end of nucleosomal double-stranded DNA (dsDNA) causes a significant increase in the distance between adjacent gyres of the duplex, and this process is not accompanied by a loss of histones; moreover, it is reversible after PARylation [24]. Such major distortions of the NCP structure may be a consequence of the ability of PARP1 to strongly interact with DNA through the DNA-binding domain (DBD), which includes three Zn-finger domains, a WGR domain, and even a BRCT domain.

Although PARP2 is the closest homolog of PARP1, its DBD is considerably different. PARP2 does not contain any known DNA-binding motifs but comprises a structure similar to the SAP motif. It also has different DNA-binding properties: a lower affinity for free DNA and compacted DNA as compared to PARP1. It has been demonstrated that during the interaction with compacted DNA, PARP2 forms a bridge between two NCPs in double-strand breaks [25]. In contrast to PARP1, the interplay between PARP2 and an NCP has not been described in much detail.

In this regard, PARP3 is less characterized compared to PARP1 and PARP2, and the processes involving PARP3 are being researched at present. PARP3 interacts with PARP1 and several DNA damage repair proteins [26,27,28]. In the cell, PARP3 is reported to be associated with several polycomb group proteins [27]. The latter finding suggests that PARP3 participates in epigenetic regulation of transcription. Notably, this enzyme does not have a structurally separate DBD. The unstructured N-terminus is responsible for this function in PARP3. Nevertheless, PARP3 is strongly activated by DNA strand breaks in vitro and can facilitate non-homologous end joining [27,28,29].

More detailed information about aspects of structural reorganization during direct binding of a PARP protein to an NCP may be obtained by single-molecule methods such as atomic force microscopy (AFM). AFM is an approach used to directly measure the geometric characteristics of individual molecules placed on mica plaque surfaces. It is one of the most widely used nano-tools for studying protein DNA complexes including NCPs [30,31,32,33,34,35,36]. This method can be employed for estimating the NCP compaction degree by measurement of the angle between DNA arms near the entry–exit site of an NCP [31,37]. Using this approach, a stabilizing effect of histone H1 on an NCP has been demonstrated [32].

In our work, we studied the interactions of PARP1, PARP2, and PARP3 with an NCP reconstituted from native core histones and Widom’s clone 603 DNA extended by 79 and 120 bp DNA arms. In particular, we determined changes in the geometric parameters of NCPs during their binding to PARP1, PARP2, or PARP3.

2. Results and Discussion

2.1. The Localization of PARP Proteins in NCP–PARP Complexes

First, we determined the site of binding of each PARP protein to our model NCP. For this purpose, reconstituted NCPs were incubated with a PARP followed by immobilization on a mica surface and visualization by AFM scanning in air. Only images of complexes containing both PARP and NCP molecules were chosen for the analysis. According to the positioning of a PARP molecule, the captured images were sorted into two categories: (i) a PARP is located close to the NCP core; (ii) the PARP is located on the linker DNA region. Figure 1 shows typical images of NCPs in their complex with PARPs. While accumulating the data, we found that each of the three PARPs presumably binds near the NCP core: in 153 out of 200 complexes for PARP1, in 148 out of 200 complexes for PARP2, and 158 out of 200 complexes for PARP3.

Figure 1.

Figure 1

Representative AFM scans of an NCP. Cores of the NCP are indicated by white arrows. PARP1, PARP2, and PARP3 molecules are pointed out by blue, green, and red arrows, respectively. (A) A PARP located close to the NCP core. (B) A PARP located on the linker DNA region.

Strong affinity (in the sub-nanomolar range) of PARP1 and PARP2 for DNA containing various structural elements has been demonstrated earlier [36,38]. In those experiments, naked DNA was used. Additionally, our previous data revealed that Kd values are almost identical when complexes of PARP1 with naked DNA and of PARP1 with an NCP are compared [39].

An earlier study uncovered a specific nature of PARP1′s binding to NCP and the ability of this protein to modulate chromatin structure through NAD+-dependent automodification without disassembly of the NCP core [40]. These authors also showed that PARP1 is associated with chromatin regions depleted of histone H1. PARP1 saturates chromatin in a molar ratio of 1:1 toward the NCP and competes with H1 for the binding to NCPs. A recent study shows the ability of PARP1 to bind DNA near the entry–exit site of an NCP through the BRCT domain of PARP1 in addition to Zn-finger domains [41]. Furthermore, a condensing effect of PARP1 binding on chromatin has been demonstrated [7]. These data are in agreement with our findings about PARP1 localization during its binding to the model NCP. Taken together, all these data may indicate a potential structural role of PARP1. The binding of PARP1 to an NCP instead of H1 in the absence of DNA damage may lead to a certain temporal pattern of chromatin alterations and to an alternative compaction degree.

Preferential binding of PARP2 to the NCP core was expected here because PARP2 possesses a significantly stronger affinity for NCP compared to naked DNA [39]. The mechanism underlying the interaction of PARP2 or PARP3 with the NCP is not clear, first of all, owing to dramatic differences in the structure of their DBDs from those of PARP1 and differences in subsequent various types of interaction with DNA [42,43]. Moreover, the interaction of PARP2 or PARP3 with the NCP in the absence of DNA damage may be mediated by core histones. In any case, the binding of PARP2 or PARP3 to an NCP may affect its geometry.

2.2. The Impact of PARP Binding on the NCP Compaction Degree

Here, we analyzed only the complexes where a PARP molecule is located close to the NCP core. We measured the angle between the linker DNAs of the NCP, i.e., the opening angle (as described in Materials and Methods), to evaluate the changes in the geometric parameters of the NCP. A similar approach was used previously [32,44].

We analyzed 200 complexes of the NCP under all conditions under study. As a reference sample in the experiment, we utilized an NCP without supplementation with any PARP. Graphical representation of the results is given in Figure 2b. The raw data are presented in Table A1, Table A2, Table A3 and Table A4. The average angle between DNA arms near the entry–exit region for NCPs in native states was estimated as 120° ± 5°. This result is consistent with the data obtained by Jan Lipfert’s group [31]. Those authors showed a dual-mode distribution in 2D density plots, which depicted a correlation between the length of unwrapped DNA and an opening-angle distribution. In contrast to their data, we did not observe such a clear-cut dual-mode distribution in our experiments (Figure A2). This discrepancy may be explained by a difference in the nucleotide sequences of the DNA used. In our work, we employed Widom’s clone 603 DNA (which is characterized by weaker affinity of binding to core histones) instead of clone 601 DNA as used in Reference [31]. The main difference between these two DNA sequences is the toughness of the NCP core: the NCP based on clone 601 DNA is tougher and therefore has less flexible DNA ends. It is probable that our model NCPs based on clone 603 DNA have insufficient differences in their opening-angle values to discriminate clearly between these two modes.

Figure 2.

Figure 2

The compaction of NCPs depending on the presence of a PARP protein. (a) Schematic representation of determined parameters of an NCP. In the image, “l1” and “l2” are DNA arm lengths, “α” is the angle between DNA arms, and “D” is the diameter of the NCP core. (b) The Gauss interpolation of the distribution of “α” angle values. The black curve: NCP samples, the blue curve: samples of NCPs supplemented with PARP1, the green curve: samples of NCPs supplemented with PARP2, and the red curve: samples of NCPs supplemented with PARP3. (c) Representation of the distribution of “α” angle values. Black bars: NCP, blue bars: NCP supplemented with PARP1, green bars: NCP supplemented with PARP2, and red bars: NCP supplemented with PARP3.

The binding of PARP1 to an NCP caused slight narrowing of the distribution of the opening arms’ angle without a significant effect on the compaction of the NCP (115° ± 4°). The difference in the measured values of the angle in the nucleosome in the presence and in the absence of PARP1 was not significant (even for a p-value of 0.9). As mentioned above, PARP1 can bind an NCP near the entry–exit site and interact with both DNA linkers. This interaction can influence the structural functioning of chromatin similarly to linker histone H1. It has been reported that the presence of H1 narrows the opening-angle distribution (meaning NCP stabilization) and does not change the compaction degree [32]. Furthermore, similarly to histone H1, PARP1 compacts chromatin, which is relaxed under PARylation conditions [7]. The authors of Reference [45] propose that the compaction is accomplished via the bringing of neighboring NCPs together by PARP1 molecules, analogously to the process observed in the polycomb group protein complex. This effect is probably due to loop formation caused by PARP1–PARP1 contacts [46]. It should be noted that the binding of PARP1 to DNA near the entry–exit site leads to the distancing of the two DNA gyres, thereby destabilizing the NCP core [24]. Thus, PARP1 loosens the NCP structure. Nonetheless, in that report, the authors demonstrated separation of fluorescent labels located on the DNA helices wrapping the histone core when PARP1 was bound. These data were obtained by the Forster resonance energy transfer technique, which does not discriminate between directions of the NCP deformation; the changes in NCP structure can occur in one of two directions: radial or axial. Because we did not detect significant changes in the compaction degree of the NCP in the presence of PARP1, the changes probably proceed in the axial direction. In this case, the influence cannot be determined by the method under study. We also cannot rule out that the previously described NCP structure distortions caused by PARP1 may affect only the DNA region that is in direct contact with the histone core. In this context, the geometry of the entry–exit site of DNA may be unaltered.

Even though PARP2 manifests significantly stronger affinity for NCPs than for naked DNA, PARP2 (just as PARP1) does not significantly affect the NCP compaction [39]. In the present work, neither the distribution of opening-angle values nor the compaction degree of the NCP was changed by the presence of PARP2 (121° ± 4°). Taking into account the standard deviation, the difference in the measured values of the angle in the nucleosome in the presence and in the absence of PARP2 was not significant (even for a p-value of 0.8). In the absence of blunt DNA ends, PARP2 probably binds to NCPs through histones. In this case, it is highly likely that PARP2 mostly binds outside the entry–exit site. Therefore, the impact on the compaction degree may be small.

Meanwhile, PARP3 exerted a distinctive effect on the compaction of the NCP core. We observed an increased compaction degree of the NCPs in the presence of PARP3 (104° ± 4°). Taking into account the standard deviation, the difference in the measured values of the angle in the nucleosome in the presence and in the absence of PARP3 was significant (a p-value of 0.001). Moreover, the presence of PARP3 induced the narrowing of the opening-angle distribution. It is worth mentioning that PARP3 is widespread in the nucleus as a part of polycomb group protein complexes. The molecular function of the polycomb group is important for homeotic gene regulation and is consequently suppressed during cell differentiation with the transition of genes into the heterochromatic state. Thus, the effect of PARP3 on NCP compaction may be required for the regulation of the access of other proteins to undamaged DNA via DNA compaction regulation.

In our work, we investigated changes in the NCP architecture during interactions with PARP1, PARP2, or PARP3 in the absence of (ADP-ribosyl)ation. The observed effects can be dramatically altered by the presence of lesions in DNA and NAD+. These alterations could also be important, especially because of the different abilities of PARP proteins to synthesize various PAR chains on an acceptor molecule, starting from the transfer of one ADP-ribose (as PARP3 does). What is more, the contribution of accompanying factors such as HPF1 could be substantial during the (ADP-ribosyl)ation and consequent NCP compaction reorganization.

Nevertheless, our study simulates the scenario where DNA is undamaged and the basic ADP-ribose transfer activity of PARPs is weak. To summarize, PARP3 is a new probable player in chromatin compaction regulation.

In conclusion, the clear difference between PARP1, PARP2, and PARP3 in their actions during this process may open up a new research field: the elucidation of PARP3′s function in chromatin compaction in the absence of DNA damage. The question is how to find the conditions (the biological process) where the observed effect is indispensable. The effect may be clarified when higher-order DNA compaction is studied in this context.

3. Materials and Methods

3.1. Reagents and Equipment

The following reagents and materials were used: 3.5 kDa cutoff dialysis membranes (Spectrum Laboratories Inc., Rancho Dominguez, CA, USA); bromophenol blue and xylene cyanol (Fluka, Buchs, Switzerland). Most of the reagents used in the study were purchased from Sigma (St. Louis, MO, USA). Recombinant Taq DNA polymerase was kindly provided by Prof. Svetlana Khodyreva (Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of Russian Academy of Sciences (ICBFM SB RAS)). Recombinant proteins—human PARP1, murine PARP2, human PARP3, and histone octamers H2A, H2B, H3, and H4 from G. gallus—were prepared and isolated as described in References [11,47,48]. AFM imaging was performed on Multimode 8 (Bruker, Billerica, MA, USA) with the help of NSG30_SS probes (TipsNano, Tallinn, Estonia). The synthesis of 1-(3-aminopropyl)-silatrane (APS) was performed as described elsewhere [49]. NCP assembly products were visualized after separation in a polyacrylamide gel by means of a Typhoon FLA 9500 system (GE Healthcare Life Science, Barrington, IL, USA) and Amersham Imager 680 (GE Healthcare Life Science, Barrington, IL, USA).

3.2. Preparation of DNA Substrates

The DNA-603-containing substrate used in the experiments was generated by PCR from a pGEM-3z/603 plasmid vector (AddGene, Watertown, MA, USA) with unique primers. The DNA construct contains 147 bp of strong positioning of Widom’s clone 603 DNA sequence surrounded by plasmid DNA sequences of 120 and 79 bp:

5′-GGGCGAATTCGAGCTCGGTACCCGGGGATCCTCTAGAGTCGGGAGCTCGGAACACTATCCGACTGGCACCGAAACGGGTACCCCAGGGACTTGAAGTAATAAGGACGGAGGGCCTCTTTCAACATCGATGCACGGTGGTTAGCCTTGGATTGCGCTCTACCGTGCGCTAAGCGTACTTAGAAGCCCGAGTGACGACTTCACACGGTAGGTGGGCGCGCGAACTGGGCACCCGAGAGTGTCGATTATTTTACGGCTCACGCTGGGGTGATTTGTACTAGGAAAACGCCTATTCGTGTATTCCGCCTTGGTCATTAGGATCCCGGACCTGCAGGCATGCAAGCTTGAG-3′.

Primer oligonucleotides 5′-GGGCGAATTCNAGCTCGGTAC-3′ and 5′-CTCAAGCTTGCATGCCTGCAG-3′ were synthesized in the Laboratory of Biomedical Chemistry at the ICBFM SB RAS (Russia). The following program in PCR was used: 3 min at 94 °C; 30 cycles of 30 s at 94 °C, 20 s at 65 °C, and 1 min at 72 °C; with final extension for 3 min at 72 °C.

After the PCR-based synthesis, the DNA substrate was purified by gel electrophoresis and isolated from the gel by the protocol from reference [50].

3.3. NCP Assembly

The NCP assembly was carried out in accordance with our previously described protocol [51]. Briefly, by quick reconstitution of NCPs in analytical amounts, the correct ratio of DNA–histones’ species was determined. Then, preparative reconstitution was performed by gradient dialysis according to the determined ratio.

3.4. Preparation of NCP Samples Containing a PARP

Sample preparation for AFM imaging was performed as described before [52]. Freshly cleaved mica was functionalized with a solution of APS for sample deposition.

The reaction mixture was composed of 10 nM NCP, NCP buffer (20 mM NaCl, 0.2 mM EDTA, 1.6 mM CHAPS, 10 mM Tris-HCl pH 7.5, and 5 mM β-mercaptoethanol) and one of PARPs at a concentration of 10 nM (PARP1), 35 nM (PARP2), or 66 nM (PARP3). The reaction mixture was incubated for 15 min at 37 °C. Then, samples were diluted tenfold with Milli-Q water and immediately deposited on the mica surface. After 120 s of deposition, the mica surface was rinsed three times with 1 mL of Milli-Q water and dried in a gentle stream of argon. The samples were stored in a desiccator before the imaging.

3.5. AFM Imaging

The visualization was performed in tapping mode in air at a tip resonance frequency of 240–440 kHz. A typical resulting image had a size of 2 µm × 2 µm at 1024 pixels/row or 4 µm × 4 µm at 2048 pixels/row. The scanning rate was either 1.0 or 0.5 Hz, respectively.

3.6. Data Analysis

All images were first processed in the Gwyddion software (http://gwyddion.net/, accessed on 1 March 2023). The ImageJ software (https://imagej.nih.gov/ij/, accessed on 1 March 2023) was employed to measure parameters of the NCP core disk and of the NCP core in complex with PARPs, the length of the NCP DNA arm, and the angle between DNA arms. The arm length was estimated by measuring the DNA from the end point to the point of “entry” into the NCP disk. Diameters of the core and core PARP were estimated as the maximal distance between two parallel tangents to the disk. The angle between NCP DNA arms was defined as an angle formed by two beams from the center of the core disc to the “entry” points of DNA arms. On the basis of the obtained data, histograms and graphs were constructed using the SigmaPlot software v.11.0 (Systat Software Inc., Chicago, IL, USA). Variances in measured values were calculated by means of Student’s t distribution with 95% confidence intervals. Measured values are shown diagrammatically in Figure 2a. When sorting PARP–NCP complexes, we chose a distance of 3 nm between the NCP core and a PARP as the border point. The resolution of the cantilever used in this work allowed us to uniquely identify a PARP separated from the NCP core when the distance was more than 3 nm. Therefore, when the proteins were located closer to the NCP core, we assumed that they were directly interacting. The workflow is illustrated in Figure A1.

Acknowledgments

We would like to thank the entire Laboratory of Bioorganic Chemistry of Enzymes (ICBFM SB RAS) for feedback on the manuscript. We acknowledge Svetlana Khodyreva for preparation of the recombinant Taq DNA polymerase.

Appendix A

Figure A1.

Figure A1

The workflow.

Figure A2.

Figure A2

The 2D density plot of wrapped length versus the opening-angle distribution of NCPs.

Table A1.

NCP parameters.

NCP # α, ° D, µm l1, µm, l2, µm
1 230 0.011 0.025 0.03
2 59 0.015 0.031 0.015
3 103 0.011 0.032 0.017
4 121 0.016 0.04 0.026
5 128 0.018 0.035 0.024
6 172 0.013 0.029 0.015
7 267 0.011 0.042 0.021
8 106 0.019 0.041 0.028
9 66 0.025 0.057 0.026
10 148 0.021 0.045 0.019
11 124 0.021 0.05 0.03
12 83 0.021 0.045 0.023
13 180 0.016 0.045 0.046
14 136 0.021 0.042 0.017
15 94 0.019 0.039 0.025
16 94 0.018 0.045 0.028
17 68 0.011 0.057 0.036
18 46 0.018 0.037 0.038
19 96 0.023 0.04 0.022
20 169 0.024 0.047 0.021
21 109 0.026 0.029 0.028
22 88 0.017 0.039 0.034
23 111 0.021 0.037 0.03
24 148 0.022 0.033 0.025
25 71 0.021 0.032 0.026
26 280 0.016 0.056 0.026
27 191 0.024 0.041 0.024
28 124 0.021 0.037 0.018
29 102 0.018 0.047 0.038
30 68 0.02 0.035 0.021
31 115 0.022 0.025 0.023
32 90 0.02 0.038 0.027
33 77 0.024 0.033 0.019
34 143 0.02 0.034 0.028
35 162 0.017 0.045 0.027
36 112 0.018 0.04 0.02
37 125 0.016 0.044 0.018
38 124 0.018 0.042 0.025
39 109 0.02 0.028 0.016
40 126 0.023 0.04 0.028
41 74 0.017 0.045 0.034
42 113 0.022 0.024 0.027
43 104 0.02 0.046 0.023
44 121 0.02 0.061 0.027
45 173 0.02 0.043 0.022
46 104 0.017 0.029 0.03
47 133 0.019 0.041 0.014
48 174 0.019 0.043 0.032
49 109 0.015 0.042 0.022
50 83 0.015 0.038 0.024
51 142 0.015 0.052 0.02
52 124 0.018 0.037 0.038
53 179 0.023 0.032 0.018
54 111 0.016 0.045 0.02
55 85 0.02 0.025 0.024
56 115 0.017 0.041 0.032
57 196 0.022 0.043 0.024
58 115 0.018 0.059 0.036
59 97 0.016 0.041 0.025
60 101 0.023 0.048 0.032
61 73 0.016 0.058 0.031
62 67 0.023 0.024 0.018
63 91 0.022 0.055 0.035
64 91 0.027 0.032 0.018
65 126 0.015 0.037 0.031
66 151 0.018 0.046 0.033
67 104 0.015 0.04 0.039
68 78 0.018 0.053 0.031
69 192 0.016 0.045 0.022
70 106 0.017 0.032 0.021
71 119 0.019 0.027 0.015
72 79 0.022 0.039 0.012
73 97 0.018 0.028 0.016
74 120 0.02 0.028 0.022
75 128 0.017 0.036 0.024
76 121 0.019 0.037 0.023
77 131 0.02 0.024 0.015
78 131 0.019 0.036 0.014
79 91 0.014 0.025 0.017
80 146 0.017 0.037 0.021
81 136 0.017 0.031 0.023
82 149 0.019 0.022 0.019
83 141 0.015 0.034 0.018
84 94 0.023 0.027 0.018
85 93 0.016 0.031 0.024
86 110 0.019 0.033 0.016
87 142 0.015 0.029 0.012
88 157 0.014 0.035 0.015
89 117 0.018 0.042 0.047
90 178 0.014 0.032 0.023
91 200 0.014 0.043 0.018
92 106 0.02 0.06 0.019
93 88 0.02 0.035 0.024
94 103 0.02 0.026 0.015
95 109 0.013 0.031 0.026
96 103 0.012 0.035 0.016
97 101 0.012 0.046 0.025
98 118 0.009 0.066 0.026
99 106 0.011 0.033 0.019
100 116 0.013 0.026 0.015
101 158 0.014 0.038 0.035
102 146 0.016 0.046 0.022
103 91 0.015 0.035 0.036
104 97 0.019 0.044 0.021
105 164 0.014 0.029 0.024
106 139 0.017 0.038 0.021
107 197 0.015 0.04 0.024
108 117 0.013 0.037 0.024
109 120 0.018 0.033 0.021
110 75 0.015 0.032 0.02
111 105 0.016 0.04 0.014
112 101 0.015 0.039 0.027
113 178 0.018 0.031 0.019
114 61 0.018 0.034 0.025
115 138 0.013 0.036 0.025
116 91 0.015 0.032 0.022
117 157 0.013 0.027 0.022
118 63 0.015 0.032 0.021
119 144 0.013 0.032 0.012
120 165 0.015 0.051 0.019
121 151 0.018 0.051 0.021
122 86 0.016 0.042 0.014
123 122 0.016 0.039 0.018
124 185 0.016 0.033 0.024
125 110 0.013 0.04 0.012
126 156 0.013 0.038 0.019
127 176 0.017 0.03 0.019
128 103 0.016 0.02 0.019
129 74 0.015 0.024 0.019
130 219 0.019 0.047 0.013
131 97 0.017 0.037 0.015
132 154 0.016 0.042 0.024
133 48 0.015 0.037 0.02
134 114 0.018 0.047 0.024
135 83 0.013 0.041 0.029
136 111 0.018 0.036 0.024
137 86 0.018 0.032 0.027
138 115 0.016 0.052 0.02
139 113 0.017 0.038 0.015
140 94 0.016 0.033 0.019
141 61 0.017 0.04 0.034
142 156 0.016 0.051 0.032
143 71 0.019 0.048 0.03
144 90 0.021 0.04 0.018
145 47 0.018 0.025 0.022
146 84 0.023 0.032 0.017
147 114 0.014 0.032 0.028
148 146 0.016 0.033 0.027
149 78 0.02 0.03 0.021
150 86 0.012 0.043 0.029
151 88 0.016 0.042 0.026
152 99 0.023 0.037 0.02
153 106 0.02 0.031 0.022
154 149 0.02 0.041 0.013
155 110 0.013 0.039 0.027
156 158 0.014 0.038 0.028
157 94 0.016 0.035 0.02
158 80 0.018 0.033 0.022
159 81 0.017 0.042 0.027
160 85 0.011 0.038 0.017
161 58 0.02 0.026 0.018
162 82 0.02 0.034 0.022
163 84 0.017 0.033 0.022
164 158 0.019 0.035 0.023
165 117 0.017 0.028 0.027
166 57 0.015 0.033 0.02
167 111 0.02 0.04 0.025
168 122 0.02 0.039 0.016
169 179 0.019 0.034 0.023
170 221 0.021 0.023 0.02
171 81 0.018 0.031 0.018
172 53 0.017 0.039 0.021
173 94 0.015 0.04 0.025
174 215 0.017 0.038 0.017
175 118 0.018 0.037 0.022
176 176 0.017 0.061 0.017
177 122 0.017 0.028 0.019
178 84 0.02 0.033 0.022
179 155 0.018 0.034 0.026
180 176 0.022 0.053 0.026
181 126 0.02 0.025 0.02
182 89 0.017 0.028 0.021
183 133 0.02 0.041 0.02
184 152 0.016 0.033 0.022
185 122 0.016 0.032 0.023
186 148 0.017 0.039 0.025
187 119 0.014 0.029 0.014
188 82 0.017 0.031 0.014
189 142 0.018 0.032 0.022
190 114 0.017 0.031 0.011
191 170 0.02 0.027 0.026
192 137 0.013 0.04 0.02
193 71 0.018 0.036 0.02
194 103 0.012 0.037 0.023
195 121 0.017 0.024 0.018
196 137 0.015 0.026 0.026
197 125 0.015 0.033 0.019
198 162 0.015 0.028 0.025
199 168 0.016 0.042 0.021
200 120 0.012 0.039 0.019

Table A2.

NCP parameters in the presence of PARP1.

NCP # α, ° D, µm l1, µm, l2, µm
1 70 0.019 0.053 0.032
2 61 0.029 0 0.027
3 128 0.026 0 0.02
4 100 0.019 0 0.014
5 61 0.023 0.031 0.03
6 96 0.024 0.035 0.019
7 117 0.032 0 0.022
8 97 0.022 0 0.016
9 109 0.023 0 0.019
10 112 0.027 0 0.026
11 120 0.027 0.016 0.033
12 107 0.033 0.023 0.035
13 179 0.023 0 0.032
14 89 0.026 0 0.018
15 58 0.026 0.03 0.021
16 176 0.024 0.019 0.017
17 112 0.024 0.008 0.019
18 112 0.019 0 0.019
19 152 0.018 0.037 0.011
20 99 0.021 0.039 0
21 98 0.022 0 0.02
22 85 0.031 0.012 0.019
23 107 0.02 0 0.024
24 107 0.022 0.033 0
25 68 0.024 0.021 0.018
26 56 0.023 0.023 0.018
27 129 0.023 0 0.02
28 97 0.026 0.008 0.032
29 131 0.023 0.04 0.02
30 85 0.02 0 0.023
31 135 0.024 0.052 0.005
32 118 0.019 0 0.028
33 162 0.024 0.003 0.023
34 107 0.027 0 0.021
35 173 0.031 0 0.022
36 122 0.024 0.04 0
37 103 0.024 0.036 0.025
38 118 0.03 0 0.024
39 98 0.025 0 0.024
40 105 0.026 0 0.024
41 161 0.023 0.044 0
42 121 0.023 0 0.02
43 114 0.023 0 0.024
44 109 0.022 0 0.025
45 85 0.032 0 0.023
46 105 0.035 0.006 0.023
47 93 0.027 0 0.021
48 96 0.023 0 0.023
49 152 0.028 0.015 0.023
50 121 0.021 0 0.015
51 116 0.032 0.031 0.004
52 153 0.024 0.015 0.02
53 87 0.023 0.031 0.008
54 74 0.03 0 0.018
55 67 0.037 0.011 0.019
56 77 0.022 0 0.02
57 76 0.021 0 0.02
58 76 0.026 0 0.02
59 0 0.025 0 0.025
60 116 0.03 0 0.019
61 101 0.033 0.035 0
62 96 0.029 0 0.019
63 145 0.028 0 0.018
64 138 0.027 0 0.027
65 101 0.024 0.022 0.019
66 108 0.023 0.016 0.015
67 220 0.02 0.044 0
68 103 0.026 0 0.02
69 102 0.024 0 0.018
70 129 0.023 0 0.021
71 84 0.018 0 0.021
72 92 0.018 0 0.016
73 98 0.022 0.016 0.023
74 147 0.017 0 0
75 82 0.028 0 0.017
76 130 0.02 0 0.016
77 179 0.029 0.043 0
78 110 0.027 0.043 0
79 130 0.026 0.042 0
80 96 0.019 0.008 0.01
81 83 0.027 0.013 0.024
82 55 0.026 0.038 0
83 144 0.021 0.041 0
84 173 0.026 0 0.018
85 149 0.02 0 0.023
86 164 0.024 0 0.028
87 69 0.026 0 0.022
88 104 0.027 0.013 0.024
89 174 0.02 0 0.023
90 171 0.024 0 0.026
91 106 0.025 0 0.011
92 83 0.021 0 0.018
93 103 0.025 0 0.016
94 120 0.024 0.022 0.021
95 200 0.023 0.033 0.024
96 140 0.023 0 0.022
97 112 0.024 0 0.019
98 168 0.021 0.016 0.02
99 132 0.019 0 0.02
100 112 0.028 0 0.016
101 128 0.024 0.037 0
102 81 0.022 0 0.018
103 63 0.025 0 0.02
104 102 0.026 0 0
105 108 0.021 0.038 0
106 91 0.021 0.039 0
107 84 0.023 0 0
108 93 0.018 0 0.014
109 141 0.021 0 0.013
110 112 0.018 0 0.025
111 75 0.02 0.019 0.019
112 91 0.018 0 0.018
113 66 0.021 0 0.022
114 93 0.026 0 0.018
115 120 0.017 0 0
116 119 0.017 0 0.029
117 142 0.022 0 0.018
118 64 0.021 0 0.026
119 78 0.018 0 0.027
120 82 0.017 0.011 0.028
121 119 0.017 0 0.033
122 85 0.018 0 0.026
123 113 0.02 0 0.025
124 138 0.022 0 0.033
125 140 0.022 0.019 0
126 117 0.017 0.012 0.02
127 97 0.02 0.017 0.025
128 87 0.016 0 0.03
129 157 0.029 0.025 0.025
130 104 0.018 0 0.023
131 63 0.025 0 0.027
132 115 0.023 0.038 0.027
133 133 0.024 0 0.026
134 98 0.029 0 0.023
135 154 0.02 0.009 0.021
136 126 0.022 0.043 0
137 59 0.018 0.037 0.008
138 133 0.018 0 0.019
139 110 0.02 0 0.02
140 132 0.016 0 0.02
141 116 0.02 0.037 0.015
142 124 0.025 0.034 0
143 98 0.014 0.043 0
144 124 0.021 0 0.023
145 111 0.022 0 0.023
146 82 0.022 0.023 0.014
147 173 0.024 0 0
148 141 0.023 0.012 0.026
149 102 0.019 0 0.026
150 115 0.021 0.044 0.009
151 122 0.021 0.007 0
152 85 0.019 0 0.025
153 97 0.021 0 0.027
154 110 0.024 0 0.022
155 137 0.022 0 0.028
156 151 0.021 0.04 0
157 92 0.026 0.04 0
158 112 0.022 0 0.023
159 54 0.014 0.035 0
160 180 0.021 0.014 0.028
161 117 0.021 0 0.018
162 77 0.022 0 0.023
163 116 0.012 0.035 0.007
164 142 0.011 0.03 0.019
165 128 0.015 0.04 0
166 75 0.012 0 0.021
167 178 0.013 0.009 0.005
168 144 0.014 0.035 0
169 155 0.015 0 0.026
170 130 0.014 0.018 0.023
171 120 0.015 0.008 0.024
172 116 0.015 0.014 0.022
173 96 0.014 0.005 0.03
174 151 0.014 0.008 0.033
175 147 0.013 0.043 0
176 69 0.019 0 0.028
177 122 0.021 0.041 0
178 122 0.019 0.036 0
179 151 0.016 0.012 0.019
180 136 0.014 0 0.023
181 148 0.016 0.037 0
182 89 0.014 0.035 0
183 173 0.014 0.009 0
184 162 0.019 0 0.025
185 120 0.021 0 0.019
186 119 0.019 0.039 0
187 85 0.024 0 0.025
188 124 0.017 0.041 0
189 118 0.017 0 0.026
190 150 0.018 0.013 0.024
191 115 0.017 0.037 0
192 90 0.017 0.033 0
193 166 0.017 0 0.028
194 108 0.02 0.035 0
195 161 0.019 0 0.028
196 171 0.016 0 0.025
197 114 0.02 0 0.017
198 87 0.019 0.037 0
199 145 0.016 0 0.027
200 125 0.02 0.011 0.028
201 110 0.021 0 0.028
202 108 0.022 0 0.018
203 74 0.014 0 0.024
204 66 0.018 0.014 0.01
205 92 0.024 0.027 0.025

Table A3.

NCP parameters in the presence of PARP2.

NCP # α, ° D, µm l1, µm, l2, µm
1 61 0.033 0 0.018
2 144 0.029 0.029 0
3 159 0.028 0.032 0.03
4 60 0.023 0.024 0.016
5 99 0.02 0 0.022
6 105 0.02 0 0.018
7 140 0.024 0 0.02
8 78 0.019 0.042 0.009
9 109 0.021 0 0
10 113 0.018 0 0.023
11 88 0.026 0.023 0.019
12 82 0.019 0 0.022
13 97 0.02 0 0.022
14 185 0.02 0 0.014
15 167 0.019 0 0.021
16 104 0.021 0 0.019
17 77 0.019 0 0.024
18 63 0.022 0 0.022
19 147 0.02 0 0.021
20 108 0.02 0.015 0
21 281 0.018 0 0.029
22 60 0.032 0 0
23 118 0.02 0 0.023
24 140 0.022 0.034 0
25 86 0.019 0 0.024
26 139 0.016 0 0.02
27 88 0.017 0 0.02
28 72 0.019 0.042 0
29 172 0.017 0 0.025
30 160 0.023 0 0.001
31 130 0.024 0.047 0.019
32 94 0.027 0 0.023
33 147 0.027 0.015 0.022
34 174 0.021 0.041 0
35 148 0.025 0 0
36 92 0.02 0.038 0.01
37 146 0.018 0.01 0.029
38 85 0.018 0 0.024
39 98 0.022 0 0.019
40 81 0.017 0.036 0
41 160 0.02 0.04 0.021
42 142 0.021 0 0.024
43 129 0.025 0.007 0.022
44 105 0.022 0.016 0.019
45 134 0.02 0 0.023
46 121 0.023 0.037 0
47 113 0.015 0 0.025
48 170 0.02 0.032 0
49 159 0.025 0.047 0
50 74 0.022 0 0.026
51 132 0.028 0 0.019
52 112 0.015 0 0.023
53 87 0.019 0 0.023
54 124 0.022 0 0.018
55 99 0.022 0 0.021
56 92 0.02 0 0.028
57 93 0.019 0 0.023
58 83 0.016 0 0
59 99 0.022 0 0.023
60 125 0.019 0 0.017
61 114 0.016 0.011 0.018
62 137 0.015 0 0.02
63 126 0.016 0.009 0.023
64 114 0.016 0.037 0
65 104 0.019 0 0.017
66 112 0.022 0 0.025
67 168 0.018 0 0.022
68 102 0.018 0 0.025
69 177 0.027 0.035 0.022
70 105 0.021 0 0.024
71 109 0.023 0 0.023
72 95 0.021 0.034 0.023
73 135 0.018 0.04 0
74 86 0.018 0 0.025
75 95 0.016 0 0.022
76 192 0.016 0 0.023
77 159 0.02 0 0.024
78 116 0.018 0 0.024
79 113 0.019 0.037 0
80 149 0.022 0.023 0.025
81 159 0.021 0 0.022
82 67 0.022 0 0.014
83 130 0.019 0 0.024
84 145 0.02 0.018 0.03
85 95 0.021 0 0.021
86 142 0.018 0.043 0
87 129 0.019 0 0.023
88 216 0.022 0 0.019
89 135 0.02 0 0.033
90 112 0.028 0.04 0.017
91 128 0.019 0.039 0.017
92 117 0.021 0.012 0.024
93 114 0.018 0.044 0
94 115 0.018 0 0.027
95 109 0.022 0.02 0.022
96 112 0.017 0 0.023
97 88 0.02 0.039 0
98 117 0.02 0.015 0.024
99 139 0.018 0 0.024
100 125 0.028 0 0.019
101 95 0.023 0.036 0
102 166 0.031 0.033 0.031
103 94 0.025 0.035 0.004
104 96 0.017 0.014 0.025
105 107 0.023 0 0.021
106 138 0.02 0.015 0.021
107 88 0.016 0 0.008
108 124 0.02 0.035 0
109 90 0.016 0 0.027
110 55 0.014 0.039 0.008
111 134 0.018 0.004 0.019
112 88 0.019 0 0.027
113 77 0.014 0 0.021
114 104 0.019 0.038 0
115 100 0.02 0.033 0.024
116 171 0.017 0 0.024
117 111 0.014 0.024 0.018
118 174 0.019 0.032 0.031
119 136 0.017 0 0.028
120 95 0.017 0.008 0.011
121 174 0.018 0.036 0
122 83 0.019 0.029 0.021
123 127 0.019 0.012 0.022
124 134 0.02 0 0.028
125 146 0.02 0 0.028
126 73 0.018 0.039 0
127 113 0.016 0 0.015
128 111 0.022 0.042 0.016
129 135 0.014 0 0.026
130 128 0.016 0.042 0
131 96 0.017 0.009 0.012
132 156 0.018 0 0.03
133 157 0.019 0 0.029
134 214 0.021 0.009 0.027
135 168 0.019 0.04 0
136 99 0.017 0 0.029
137 83 0.02 0 0.024
138 123 0.015 0.036 0
139 114 0.017 0 0.032
140 121 0.021 0 0.028
141 144 0.018 0 0.025
142 97 0.018 0.04 0
143 143 0.018 0.036 0
144 104 0.02 0.038 0.019
145 99 0.018 0 0.027
146 85 0.019 0 0.02
147 103 0.017 0 0.027
148 143 0.017 0.045 0
149 133 0.016 0 0.032
150 111 0.016 0.006 0.029
151 153 0.019 0.013 0.03
152 109 0.019 0.008 0.028
153 128 0.014 0 0.032
154 93 0.021 0.007 0.029
155 99 0.02 0.04 0.01
156 120 0.017 0.035 0.007
157 96 0.017 0.038 0.002
158 111 0.014 0.038 0
159 122 0.017 0.011 0.028
160 133 0.014 0.008 0.028
161 113 0.021 0 0.028
162 73 0.015 0.004 0.03
163 113 0.018 0.01 0.032
164 97 0.019 0.04 0
165 86 0.017 0.038 0
166 142 0.017 0.007 0.029
167 77 0.019 0.007 0.028
168 78 0.018 0.007 0.019
169 154 0.015 0 0.027
170 128 0.02 0 0.031
171 125 0.02 0.042 0
172 101 0.018 0.01 0.023
173 109 0.017 0.037 0
174 86 0.017 0 0.026
175 103 0.015 0 0.031
176 142 0.02 0.01 0
177 102 0.02 0 0.029
178 152 0.018 0 0.027
179 172 0.016 0 0.025
180 118 0.019 0 0.028
181 148 0.019 0 0.03
182 102 0.016 0 0.026
183 84 0.021 0.037 0.013
184 145 0.015 0 0.031
185 95 0.014 0 0.024
186 144 0.015 0.033 0
187 102 0.02 0.007 0.018
188 217 0.016 0.048 0
189 172 0.015 0 0
190 145 0.018 0.04 0
191 162 0.018 0.006 0
192 107 0.022 0.005 0.025
193 123 0.014 0.041 0
194 84 0.018 0.037 0
195 267 0.019 0.049 0.029
196 114 0.017 0.039 0
197 96 0.016 0 0.024
198 91 0.019 0 0.023
199 148 0.017 0 0.008
200 136 0.019 0.033 0.005
201 85 0.019 0.039 0

Table A4.

NCP parameters in the presence of PARP3.

NCP # α, ° D, µm l1, µm, l2, µm
1 106 0.025 0.005 0.025
2 156 0.024 0 0.024
3 87 0.02 0 0.02
4 77 0.024 0 0.025
5 98 0.025 0 0.022
6 77 0.015 0 0.027
7 81 0.017 0 0.024
8 138 0.02 0.041 0.01
9 83 0.018 0 0.016
10 102 0.021 0.035 0.009
11 93 0.016 0 0.027
12 95 0.013 0.035 0
13 119 0.019 0 0.021
14 174 0.021 0 0.021
15 122 0.017 0 0.023
16 97 0.013 0.04 0
17 84 0.018 0.037 0
18 75 0.02 0.043 0
19 84 0.024 0 0.024
20 96 0.018 0 0.028
21 145 0.015 0 0.023
22 69 0.02 0 0.024
23 81 0.018 0 0.028
24 103 0.018 0 0.021
25 79 0.018 0.041 0
26 154 0.019 0 0.032
27 99 0.018 0 0.023
28 106 0.015 0 0.024
29 116 0.018 0 0.023
30 87 0.021 0 0
31 98 0.018 0.041 0
32 134 0.019 0 0.021
33 111 0.02 0.038 0
34 104 0.018 0 0.026
35 161 0.016 0 0.023
36 109 0.017 0 0.027
37 114 0.015 0 0.028
38 110 0.018 0 0.027
39 116 0.019 0 0.021
40 101 0.014 0.039 0
41 75 0.012 0 0.023
42 135 0.018 0.034 0
43 83 0.017 0 0.023
44 94 0.014 0 0.02
45 87 0.019 0 0.023
46 157 0.016 0.042 0.015
47 81 0.019 0 0.03
48 62 0.02 0 0.032
49 95 0.02 0 0.022
50 132 0.018 0.038 0
51 108 0.017 0.038 0
52 71 0.015 0 0.024
53 140 0.017 0 0.021
54 99 0.019 0 0.014
55 45 0.017 0 0.024
56 94 0.019 0 0.024
57 119 0.014 0 0.023
58 116 0.018 0 0.021
59 38 0.019 0 0.018
60 74 0.016 0 0.023
61 53 0.015 0.037 0
62 95 0.018 0 0.02
63 166 0.014 0.015 0.021
64 61 0.015 0 0.019
65 122 0.018 0 0.024
66 209 0.016 0 0.023
67 119 0.015 0 0.023
68 78 0.014 0 0.023
69 121 0.012 0.037 0
70 118 0.017 0 0
71 101 0.017 0.04 0.01
72 105 0.018 0.027 0.022
73 87 0.016 0.009 0.024
74 66 0.016 0 0.018
75 178 0.017 0.038 0.014
76 161 0.026 0.016 0.023
77 97 0.016 0.018 0.024
78 82 0.018 0 0.024
79 142 0.016 0.035 0
80 123 0.016 0 0.026
81 82 0.015 0 0.023
82 133 0.013 0 0.022
83 71 0.017 0 0.024
84 93 0.019 0.008 0.022
85 105 0.021 0 0.024
86 98 0.016 0 0.028
87 144 0.014 0 0.028
88 113 0.02 0.007 0.021
89 124 0.015 0 0.027
90 107 0.016 0 0.022
91 85 0.016 0 0.026
92 154 0.018 0.011 0.028
93 119 0.015 0 0.019
94 90 0.019 0 0.021
95 106 0.018 0 0.024
96 87 0.017 0.04 0
97 60 0.017 0.038 0.011
98 83 0.014 0.038 0
99 119 0.017 0 0.022
100 118 0.014 0 0.023
101 89 0.014 0 0.025
102 99 0.016 0.004 0.025
103 131 0.017 0 0.02
104 118 0.015 0 0.021
105 102 0.015 0 0
106 68 0.015 0.009 0.025
107 86 0.013 0 0.026
108 82 0.016 0.017 0.022
109 104 0.016 0 0.022
110 119 0.015 0.039 0.004
111 74 0.013 0.01 0.026
112 100 0.017 0.008 0.003
113 96 0.017 0 0.02
114 89 0.012 0.038 0
115 117 0.015 0.038 0
116 105 0.014 0 0.024
117 59 0.016 0 0.028
118 151 0.013 0 0.03
119 115 0.013 0.005 0.028
120 83 0.017 0 0.006
121 169 0.014 0.019 0.031
122 130 0.02 0.043 0.004
123 135 0.015 0.034 0
124 110 0.012 0.008 0.017
125 122 0.016 0 0.026
126 105 0.019 0.013 0
127 96 0.014 0.012 0.02
128 93 0.018 0 0.026
129 132 0.018 0 0.027
130 85 0.018 0 0.024
131 85 0.015 0 0.03
132 93 0.016 0.008 0.026
133 99 0.02 0 0.028
134 97 0.015 0.039 0
135 123 0.02 0 0.021
136 95 0.013 0 0.029
137 97 0.018 0.04 0.006
138 119 0.014 0.038 0
139 93 0.016 0 0.024
140 93 0.017 0.013 0.017
141 97 0.017 0.033 0.006
142 109 0.017 0 0.023
143 98 0.016 0 0.021
144 113 0.022 0.026 0.027
145 79 0.018 0 0.026
146 134 0.023 0 0
147 117 0.021 0 0.026
148 60 0.016 0.035 0
149 86 0.02 0.009 0.024
150 111 0.019 0.034 0
151 148 0.018 0 0.024
152 45 0.017 0 0.031
153 92 0.017 0.038 0
154 93 0.017 0 0.024
155 99 0.021 0.042 0.015
156 93 0.022 0 0.031
157 114 0.018 0.015 0.028
158 99 0.023 0.019 0.027
159 89 0.018 0.008 0.022
160 140 0.021 0 0.034
161 87 0.017 0.016 0.025
162 94 0.019 0.037 0
163 108 0.02 0 0.025
164 89 0.02 0.032 0
165 86 0.022 0.006 0.028
166 100 0.021 0.009 0.029
167 75 0.019 0.038 0
168 52 0.02 0.037 0
169 114 0.018 0 0.027
170 97 0.018 0.04 0
171 91 0.017 0.013 0.024
172 81 0.021 0.045 0
173 152 0.027 0.031 0
174 91 0.018 0 0.028
175 170 0.019 0.017 0.033
176 55 0.02 0.006 0.024
177 88 0.017 0 0.026
178 81 0.023 0 0.029
179 59 0.018 0 0
180 138 0.017 0.037 0
181 74 0.017 0 0.023
182 75 0.019 0.036 0
183 82 0.02 0 0.024
184 102 0.023 0.016 0.004
185 170 0.02 0.044 0
186 113 0.017 0 0.023
187 71 0.021 0.039 0
188 157 0.019 0 0.027
189 156 0.018 0.038 0
190 170 0.018 0 0.006
191 85 0.022 0.042 0.006
192 104 0.015 0 0.021
193 175 0.021 0.04 0.009
194 117 0.019 0.038 0.014
195 56 0.018 0.042 0
196 126 0.015 0 0.027
197 99 0.019 0 0.032
198 96 0.015 0 0.022
199 98 0.018 0.009 0.023
200 65 0.021 0.042 0
201 70 0.026 0 0.029

Author Contributions

Conceptualization, M.K. and E.B.; methodology and formal analysis, A.U. and M.J.; validation, V.G. and A.L.; writing—original draft preparation, M.K.; writing—review and editing, O.L. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data needed to reproduce our results are contained within the article. Raw data are available upon request.

Conflicts of Interest

The authors declare no conflict of interest.

Funding Statement

This research was funded by the Russian Science Foundation (project No. 22-74-10059). The purification of PARP3 was supported by the Russian state-funded project for ICBFM SB RAS (grant number 121031300041-4).

Footnotes

Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

References

  • 1.Rao S.S., Huntley M.H., Durand N.C., Stamenova E.K., Bochkov I.D., Robinson J.T., Sanborn A.L., Machol I., Omer A.D., Lander E.S., et al. A 3D map of the human genome at kilobase resolution reveals principles of chromatin looping. Cell. 2014;159:1665–1680. doi: 10.1016/j.cell.2014.11.021. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Kouzarides T. Chromatin modifications and their function. Cell. 2007;128:693–705. doi: 10.1016/j.cell.2007.02.005. [DOI] [PubMed] [Google Scholar]
  • 3.McGinty R.K., Tan S. Nucleosome structure and function. Chem. Rev. 2015;115:2255–2273. doi: 10.1021/cr500373h. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Öztürk M.A., De M., Cojocaru V., Wade R.C. Chromatosome Structure and Dynamics from Molecular Simulations. Annu. Rev. Phys. Chem. 2020;71:101–119. doi: 10.1146/annurev-physchem-071119-040043. [DOI] [PubMed] [Google Scholar]
  • 5.Lyubchenko Y.L. Centromere chromatin: A loose grip on the nucleosome? Nat. Struct. Mol. Biol. 2014;21:8. doi: 10.1038/nsmb.2745. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Stormberg T., Lyubchenko Y.L. The Sequence Dependent Nanoscale Structure of CENP-A Nucleosomes. Int. J. Mol. Sci. 2022;23:11385. doi: 10.3390/ijms231911385. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Wacker D.A., Ruhl D.D., Balagamwala E.H., Hope K.M., Zhang T., Kraus W.L. The DNA binding and catalytic domains of poly(ADP-ribose) polymerase 1 cooperate in the regulation of chromatin structure and transcription. Mol. Cell. Biol. 2007;27:7475–7485. doi: 10.1128/MCB.01314-07. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Lüscher B., Ahel I., Altmeyer M., Ashworth A., Bai P., Chang P., Cohen M., Corda D., Dantzer F., Daugherty M.D., et al. ADP-ribosyltransferases, an update on function and nomenclature. FEBS J. 2022;289:7399–7410. doi: 10.1111/febs.16142. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Talhaoui I., Lebedeva N.A., Zarkovic G., Saint-Pierre C., Kutuzov M.M., Sukhanova M.V., Matkarimov B.T., Gasparutto D., Saparbaev M.K., Lavrik O.I., et al. Poly(ADP-ribose) polymerases covalently modify strand break termini in DNA fragments in vitro. Nucleic Acids Res. 2016;44:9279–9295. doi: 10.1093/nar/gkw675. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Zarkovic G., Belousova E.A., Talhaoui I., Saint-Pierre C., Kutuzov M.M., Matkarimov B.T., Biard D., Gasparutto D., Lavrik O.I., Ishchenko A.A. Characterization of DNA ADP-ribosyltransferase activities of PARP2 and PARP3: New insights into DNA ADP-ribosylation. Nucleic Acids Res. 2018;46:2417–2431. doi: 10.1093/nar/gkx1318. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Belousova E.A., Ishchenko A.A., Lavrik O.I. Dna is a New Target of Parp3. Sci. Rep. 2018;8:4176. doi: 10.1038/s41598-018-22673-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Vyas S., Matic I., Uchima L., Rood J., Zaja R., Hay R.T., Ahel I., Chang P. Family-wide analysis of poly(ADP-ribose) polymerase activity. Nat. Commun. 2014;5:4426. doi: 10.1038/ncomms5426. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Kutuzov M.M., Belousova E.A., Ilina E.S., Lavrik O.I. Impact of PARP1, PARP2 & PARP3 on the Base Excision Repair of Nucleosomal DNA. Adv. Exp. Med. Biol. 2020;1241:47–57. doi: 10.1007/978-3-030-41283-8_4. [DOI] [PubMed] [Google Scholar]
  • 14.Ray Chaudhuri A., Nussenzweig A. The multifaceted roles of PARP1 in DNA repair and chromatin remodelling. Nat. Rev. Mol. Cell. Biol. 2017;18:610–621. doi: 10.1038/nrm.2017.53. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Alemasova E.E., Lavrik O.I. Poly(ADP-ribosyl)ation by PARP1: Reaction mechanism and regulatory proteins. Nucleic Acids Res. 2019;47:3811–3827. doi: 10.1093/nar/gkz120. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Van Beek L., McClay É., Patel S., Schimpl M., Spagnolo L., Maia de Oliveira T. PARP Power: A Structural Perspective on PARP1, PARP2, and PARP3 in DNA Damage Repair and Nucleosome Remodelling. Int. J. Mol. Sci. 2021;22:5112. doi: 10.3390/ijms22105112. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Beck C., Robert I., Reina-San-Martin B., Schreiber V., Dantzer F. Poly(ADP-ribose) polymerases in double-strand break repair: Focus on PARP1, PARP2 and PARP3. Exp. Cell. Res. 2014;329:18–25. doi: 10.1016/j.yexcr.2014.07.003. [DOI] [PubMed] [Google Scholar]
  • 18.Virág L., Robaszkiewicz A., Rodriguez-Vargas J.M., Oliver F.J. Poly(ADP-ribose) signaling in cell death. Mol. Aspects Med. 2013;34:1153–1167. doi: 10.1016/j.mam.2013.01.007. [DOI] [PubMed] [Google Scholar]
  • 19.Belousova E.A., Lavrik O.I. The Role of PARP1 and PAR in ATP-Independent Nucleosome Reorganisation during the DNA Damage Response. Genes. 2022;14:112. doi: 10.3390/genes14010112. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Poirier G.G., de Murcia G., Jongstra-Bilen J., Niedergang C., Mandel P. Poly(ADP-ribosyl)ation of polynucleosomes causes relaxation of chromatin structure. Proc. Natl. Acad. Sci. USA. 1982;79:3423–3427. doi: 10.1073/pnas.79.11.3423. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Gibbs-Seymour I., Fontana P., Rack J.G.M., Ahel I. HPF1/C4orf27 Is a PARP-1-Interacting Protein that Regulates PARP-1 ADP-Ribosylation Activity. Mol. Cell. 2016;62:432–442. doi: 10.1016/j.molcel.2016.03.008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Suskiewicz M.J., Zobel F., Ogden T.E.H., Fontana P., Ariza A., Yang J.C., Zhu K., Bracken L., Hawthorne W.J., Ahel D., et al. HPF1 completes the PARP active site for DNA damage-induced ADP-ribosylation. Nature. 2020;579:598–602. doi: 10.1038/s41586-020-2013-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Longarini E.J., Matic I. The fast-growing business of Serine ADP-ribosylation. DNA Repair. 2022;118:103382. doi: 10.1016/j.dnarep.2022.103382. [DOI] [PubMed] [Google Scholar]
  • 24.Sultanov D.C., Gerasimova N.S., Kudryashova K.S., Maluchenko N.V., Kotova E.Y., Langelier M.F., Pascal J.M., Kirpichnikov M.P., Feofanov A.V., Studitsky V.M. Unfolding of core nucleosomes by PARP-1 revealed by spFRET microscopy. AIMS Genet. 2017;4:21–31. doi: 10.3934/genet.2017.1.21. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Gaullier G., Roberts G., Muthurajan U.M., Bowerman S., Rudolph J., Mahadevan J., Jha A., Rae P.S., Luger K. Bridging of nucleosome-proximal DNA double-strand breaks by PARP2 enhances its interaction with HPF1. PLoS ONE. 2020;15:e0240932. doi: 10.1371/journal.pone.0240932. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Loseva O., Jemth A.S., Bryant H.E., Schüler H., Lehtiö L., Karlberg T., Helleday T. PARP-3 is a mono-ADP-ribosylase that activates PARP-1 in the absence of DNA. J. Biol. Chem. 2010;285:8054–8060. doi: 10.1074/jbc.M109.077834. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Rouleau M., McDonald D., Gagné P., Ouellet M.E., Droit A., Hunter J.M., Dutertre S., Prigent C., Hendzel M.J., Poirier G.G. PARP-3 associates with polycomb group bodies and with components of the DNA damage repair machinery. J. Cell Biochem. 2007;100:385–401. doi: 10.1002/jcb.21051. [DOI] [PubMed] [Google Scholar]
  • 28.Rulten S.L., Fisher A.E., Robert I., Zuma M.C., Rouleau M., Ju L., Poirier G., Reina-San-Martin B., Caldecott K.W. PARP-3 and APLF function together to accelerate nonhomologous end-joining. Mol. Cell. 2011;41:33–45. doi: 10.1016/j.molcel.2010.12.006. [DOI] [PubMed] [Google Scholar]
  • 29.Augustin A., Spenlehauer C., Dumond H., Ménissier-De Murcia J., Piel M., Schmit A.C., Apiou F., Vonesch J.L., Kock M., Bornens M., et al. PARP-3 localizes preferentially to the daughter centriole and interferes with the G1/S cell cycle progression. J. Cell Sci. 2003;116:1551–1562. doi: 10.1242/jcs.00341. [DOI] [PubMed] [Google Scholar]
  • 30.Sun Z., Stormberg T., Filliaux S., Lyubchenko Y.L. Three-Way DNA Junction as an End Label for DNA in Atomic Force Microscopy Studies. Int. J. Mol. Sci. 2022;23:11404. doi: 10.3390/ijms231911404. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Konrad S.F., Vanderlinden W., Lipfert J. Quantifying epigenetic modulation of nucleosome breathing by high-throughput AFM imaging. Biophys. J. 2022;121:841–851. doi: 10.1016/j.bpj.2022.01.014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Würtz M., Aumiller D., Gundelwein L., Jung P., Schütz C., Lehmann K., Tóth K., Rohr K. DNA accessibility of chromatosomes quantified by automated image analysis of AFM data. Sci. Rep. 2019;9:12788. doi: 10.1038/s41598-019-49163-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Lyubchenko Y.L., Shlyakhtenko L.S. AFM for analysis of structure and dynamics of DNA and protein-DNA complexes. Methods. 2009;47:206–213. doi: 10.1016/j.ymeth.2008.09.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Shlyakhtenko L.S., Lushnikov A.Y., Lyubchenko Y.L. Dynamics of nucleosomes revealed by time-lapse atomic force microscopy. Biochemistry. 2009;48:7842–7848. doi: 10.1021/bi900977t. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Sukhanova M.V., Abrakhi S., Joshi V., Pastre D., Kutuzov M.M., Anarbaev R.O., Curmi P.A., Hamon L., Lavrik O.I. Single molecule detection of PARP1 and PARP2 interaction with DNA strand breaks and their poly(ADP-ribosyl)ation using high-resolution AFM imaging. Nucleic Acids Res. 2016;44:e60. doi: 10.1093/nar/gkv1476. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Sukhanova M.V., Hamon L., Kutuzov M.M., Joshi V., Abrakhi S., Dobra I., Curmi P.A., Pastre D., Lavrik O.I. A Single-Molecule Atomic Force Microscopy Study of PARP1 and PARP2 Recognition of Base Excision Repair DNA Intermediates. J. Mol. Biol. 2019;431:2655–2673. doi: 10.1016/j.jmb.2019.05.028. [DOI] [PubMed] [Google Scholar]
  • 37.Filenko N.A., Kolar C., West J.T., Smith S.A., Hassan Y.I., Borgstahl G.E., Zempleni J., Lyubchenko Y.L. The role of histone H4 biotinylation in the structure of nucleosomes. PLoS ONE. 2011;6:e16299. doi: 10.1371/journal.pone.0016299. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Kutuzov M.M., Khodyreva S.N., Amé J.C., Ilina E.S., Sukhanova M.V., Schreiber V., Lavrik O.I. Interaction of PARP-2 with DNA structures mimicking DNA repair intermediates and consequences on activity of base excision repair proteins. Biochimie. 2013;95:1208–1215. doi: 10.1016/j.biochi.2013.01.007. [DOI] [PubMed] [Google Scholar]
  • 39.Kutuzov M.M., Belousova E.A., Kurgina T.A., Ukraintsev A.A., Vasil’eva I.A., Khodyreva S.N., Lavrik O.I. The contribution of PARP1, PARP2 and poly(ADP-ribosyl)ation to base excision repair in the nucleosomal context. Sci. Rep. 2021;11:4849. doi: 10.1038/s41598-021-84351-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Kim M.Y., Mauro S., Gévry N., Lis J.T., Kraus W.L. NAD+-dependent modulation of chromatin structure and transcription by nucleosome binding properties of PARP-1. Cell. 2004;119:803–814. doi: 10.1016/j.cell.2004.11.002. [DOI] [PubMed] [Google Scholar]
  • 41.Rudolph J., Muthurajan U.M., Palacio M., Mahadevan J., Roberts G., Erbse A.H., Dyer P.N., Luger K. The BRCT domain of PARP1 binds intact DNA and mediates intrastrand transfer. Mol. Cell. 2021;81:4994–5006. doi: 10.1016/j.molcel.2021.11.014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Grundy G.J., Polo L.M., Zeng Z., Rulten S.L., Hoch N.C., Paomephan P., Xu Y., Sweet S.M., Thorne A.W., Oliver A.W., et al. PARP3 is a sensor of nicked nucleosomes and monoribosylates histone H2B(Glu2) Nat. Commun. 2016;7:12404. doi: 10.1038/ncomms12404. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Obaji E., Maksimainen M.M., Galera-Prat A., Lehtiö L. Activation of PARP2/ARTD2 by DNA damage induces conformational changes relieving enzyme autoinhibition. Nat. Commun. 2021;12:3479. doi: 10.1038/s41467-021-23800-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Stumme-Diers M.P., Banerjee S., Hashemi M., Sun Z., Lyubchenko Y.L. Nanoscale dynamics of centromere nucleosomes and the critical roles of CENP-A. Nucleic Acids Res. 2018;46:94–103. doi: 10.1093/nar/gkx933. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.Francis N.J., Kingston R.E., Woodcock C.L. Chromatin compaction by a polycomb group protein complex. Science. 2004;306:1574–1577. doi: 10.1126/science.1100576. [DOI] [PubMed] [Google Scholar]
  • 46.Bell N.A.W., Haynes P.J., Brunner K., de Oliveira T.M., Flocco M.M., Hoogenboom B.W., Molloy J.E. Single-molecule measurements reveal that PARP1 condenses DNA by loop stabilization. Sci. Adv. 2021;7:eabf3641. doi: 10.1126/sciadv.abf3641. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.Amé J.C., Kalisch T., Dantzer F., Schreiber V. Purification of recombinant poly(ADP-ribose) polymerases. Methods Mol. Biol. 2011;780:135–152. doi: 10.1007/978-1-61779-270-0_9. [DOI] [PubMed] [Google Scholar]
  • 48.Peterson C.L., Hansen J.C. Chicken erythrocyte histone octamer preparation. Cold Spring Harb. Protoc. 2008;2008:prot5112. doi: 10.1101/pdb.prot5112. [DOI] [PubMed] [Google Scholar]
  • 49.Shlyakhtenko L.S., Gall A.A., Lyubchenko Y.L. Mica functionalization for imaging of DNA and protein-DNA complexes with atomic force microscopy. Methods Mol. Biol. 2013;931:295–312. doi: 10.1007/978-1-62703-056-4_14. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 50.Green M.R., Sambrook J. Isolation of DNA Fragments from Polyacrylamide Gels by the Crush and Soak Method. Cold Spring Harb. Protoc. 2019;2019:prot100479. doi: 10.1101/pdb.prot100479. [DOI] [PubMed] [Google Scholar]
  • 51.Kutuzov M.M., Kurgina T.A., Belousova E.A., Khodyreva S.N., Lavrik O.I. Optimization of nucleosome assembling from histones and model DNAs and estimation of the reconstitution efficiency. Biopolym. Cells. 2019;35:91–98. doi: 10.7124/bc.00099A. [DOI] [Google Scholar]
  • 52.Stormberg T., Stumme-Diers M., Lyubchenko Y.L. Sequence-dependent nucleosome nanoscale structure characterized by atomic force microscopy. FASEB J. 2019;33:10916–10923. doi: 10.1096/fj.201901094R. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

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Data Availability Statement

The data needed to reproduce our results are contained within the article. Raw data are available upon request.


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