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Assembly and cryo-EM of PARP2–HPF1 bound to mononucleosomes a, b, SDS–PAGE (a) and native gel (b) showing the PARP2–HPF1–nucleosome complex assembly for cryo-EM. Note the shift in the PARP2–nucleosome complex migration upon binding of HPF1 in b. c, SDS–PAGE and immunoblotting showing PARP2 PARylation of nucleosomes. HPF1 is required for H3 PARylation. d, Representative cryo-EM micrograph collected with Titan Krios electron microscope at 300 keV. Bridging of two nucleosomes by PARP2–HPF1 is clearly visible in the raw data. Complex particles in multiple orientations are visible. e, Representative 2D class averages showing two nucleosomes bridged by PARP2–HPF1. Two nucleosomes are positioned in an almost perpendicular orientation. PARP2–HPF1 density between two nucleosomes is clearly visible. Many details in nucleosomes are visible in 2D class averages. For gel source data, see Supplementary Fig. 1.
Source publication
Breaks in DNA strands recruit the protein PARP1 and its paralogue PARP2 to modify histones and other substrates through the addition of mono- and poly(ADP-ribose) (PAR)1–5. In the DNA damage responses, this post-translational modification occurs predominantly on serine residues6–8 and requires HPF1, an accessory factor that switches the amino acid...
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Poly(ADP‐ribosyl)ation is predominantly catalyzed by Poly(ADP‐ribose) polymerase 1 (PARP1) in response to DNA damage, mediating the DNA repair process to maintain genomic integrity. Single‐strand (SSB) and double‐strand (DSB) DNA breaks are bona fide stimulators of PARP1 activity. However, PAR‐mediated PARP1 regulation remains unexplored. Here, we...
Poly(ADP-ribose) Polymerase 2 (PARP2) is one of three DNA-dependent PARPs involved in the detection of DNA damage. Upon binding to DNA double-strand breaks, PARP2 uses nicotinamide adenine dinucleotide to synthesize poly(ADP-ribose) (PAR) onto itself and other proteins, including histones. PAR chains in turn promote the DNA damage response by recru...
Posttranslational modifications exist in different varieties to regulate diverse characteristics of their substrates, ultimately leading to maintenance of cell health. The enzymes of the intracellular PARP family can transfer either a single ADP-ribose to targets, in a reaction called mono(ADP-ribosyl)ation or MARylation, or multiple to form chains...
Purpose:
Poly(ADP-ribose) polymerase 1 (PARP1) is necessary for single-strand break (SSB) repair by sensing DNA breaks and facilitating DNA repair through poly ADP-ribosylation of several DNA-binding and repair proteins. Inhibition of PARP1 results in collapsed DNA replication fork and double-strand breaks (DSBs). Accumulation of DSBs goes beyond...
Hepatocellular carcinoma (HCC) is a major global health concern, representing one of the leading causes of cancer-related deaths. Despite various treatment options, the prognosis for HCC patients remains poor, emphasizing the need for a deeper understanding of the factors contributing to HCC development. This study investigates the role of poly(ADP...
Citations
... In PARP2, the WGR domain is responsible for DNA binding and has been shown to bridge over doublestrand DNA gaps [13]. Interaction of the WGR domain with DNA triggers structural changes in the HD domain, leading to a shift of the catalytic domain and opening of the active site [14,15]. The role of PARP1/2 in DNA lesion detection has gained extensive interest upon the discovery of sensitivity of cells defective in homologous recombination to their inhibition. ...
Human diphtheria toxin-like ADP-ribosyltransferases, PARPs and tankyrases, transfer ADP-ribosyl groups to other macromolecules, thereby controlling various signaling events in cells. They are considered promising drug targets, especially in oncology, and some small molecule inhibitors have already been developed. These inhibitors typically interact with the nicotinamide binding site and extend along the NAD ⁺ binding groove of the catalytic domain. Quinazolin-4-ones have been explored as promising scaffolds for such inhibitors and we have identified a new position within the catalytic domain that has not been extensively studied yet. In this study, we investigate larger substituents at the C-8 position and, using X-ray crystallography, we demonstrate that nitro- and diol-substituents engage in new interactions with TNKS2, improving both affinity and selectivity. Both nitro- and diol-substituents exhibit intriguing inhibition of TNKS2, with compound 49 displaying an IC 50 of 65 nM, while compound 40 ’s IC 50 value is 14 nM. Both analogues show efficacy in cell assays and attenuate the tankyrase-controlled Wnt/β-catenin signaling with sub-micromolar IC 50 . When tested against a wider panel of enzymes, compound 40 displayed high selectivity towards tankyrases, whereas 49 also inhibited other PARPs. The results offer new insights for inhibitor development targeting tankyrases and PARPs by focusing on the subsite between a mobile active site loop and the canonical nicotinamide binding site.
... Indeed, we found that PARP1/HPF1 initially installs PAR chains predominantly on the double-strand break-proximal side of nucleosomes (Fig. 1). This result is consistent with a structure of PARP2 and HPF1 bound to a nucleosome 66 , which suggests that the H3 and H2B tails closest to the active site are likely to be favored for modification. ...
The chromatin remodeler ALC1 is activated by DNA damage-induced poly(ADP-ribose) deposited by PARP1/PARP2 and their co-factor HPF1. ALC1 has emerged as a cancer drug target, but how it is recruited to ADP-ribosylated nucleosomes to affect their positioning near DNA breaks is unknown. Here we find that PARP1/HPF1 preferentially initiates ADP-ribosylation on the histone H2B tail closest to the DNA break. To dissect the consequences of such asymmetry, we generate nucleosomes with a defined ADP-ribosylated H2B tail on one side only. The cryo-electron microscopy structure of ALC1 bound to such an asymmetric nucleosome indicates preferential engagement on one side. Using single-molecule FRET, we demonstrate that this asymmetric recruitment gives rise to directed sliding away from the DNA linker closest to the ADP-ribosylation site. Our data suggest a mechanism by which ALC1 slides nucleosomes away from a DNA break to render it more accessible to repair factors.
... The research offered novel perspectives on aerobic glycolysis in rapid-proliferating cells. Furthermore, NAD + -consuming enzymes (NADases) directly or indirectly impact DNA repair and gene transcription (2,3). ...
Nicotinamide adenine dinucleotide (NAD⁺) is indispensable for various oxidation-reduction reactions in mammalian cells, particularly during energy production. Malignant cells increase the expression levels of NAD⁺ biosynthesis enzymes for rapid proliferation and biomass production. Furthermore, mounting proof has indicated that NAD-degrading enzymes (NADases) play a role in creating the immunosuppressive tumor microenvironment (TME). Interestingly, both inhibiting NAD⁺ synthesis and targeting NADase have positive implications for cancer treatment. Here we summarize the detrimental outcomes of increased NAD⁺ production, the functions of NAD⁺ metabolic enzymes in creating an immunosuppressive TME, and discuss the progress and clinical translational potential of inhibitors for NAD⁺ synthesis and therapies targeting NADase.
... The action produced by this enzyme in the damaged DNA plays an important role, activating the repair pathways in a single chain or breaking both DNA chains (Murai et al., 2012). In response to the damage caused in the DNA, the PAR post-translational modification mainly takes place in the serine amino acids, involving the participation of the HPF1 factor, which changes the specificity of the glutamate residue left by the serine in PARP2 (Bilokapic et al., 2020). Different PARP2 inhibitors (such as olaparib, veliparib, and MK-4827) are found in the advanced stage of clinical trials. ...
Objective: To analyze the molecular docking of secondary metabolites of soursop on the enzyme markers of breast cancer. Design/Methodology/Approach: Crystals of PARP2 and PRMT5 enzymes were obtained from RCSB-PDB. Both crystals were processed using bioinformatic tools (e.g., SWISS-MODEL, UCSF-Chimera, and ScanProsite), prior to molecular docking and dynamics. The Annona muricata L. metabolites were obtained from Pubchem for their use in several in silico analysis. The Autodock algorithm was used to obtain the molecular docking. Once the most stable conformations were obtained for the ligands of each enzyme, their complexes were subjected to 10 ns of molecular dynamics using GROMACS. Meanwhile, the HPF1-PARP2 and the MEP50-PRMT5 heterodimeric interactions were carried out using the HDOCK server. Finally, the possible biotransformation reactions were studied using QSAR models. Results: The kaempferol-3-O-rutinoside metabolite showed potential biopharmaceutical use as an inhibitor of the PARP2 enzyme. The coreximin ligand showed potential biopharmaceutical use as an inhibitor of the PRMT5 enzyme. The inhibitor impacted the PRMT5-MEP50 interaction. The QSAR models indicated that methylation, O-glucuronidation, and O-dealkylation were the most likely biotransformation reactions among the metabolites with the highest degree of inhibition. Study Limitation/Implications: in silico analysis on inhibition of key proteins. Findings/Conclusions: The kaempferol-3-O-rutinoside chemical compound showed potential as a PARP2 inhibitor. The coreximin chemical compound showed potential as a PRMT5 inhibitor. The protein-protein interaction between PRMT5 and MEP50 was impacted by the inhibitor; however, this was not the case with the PARP2 enzyme.
... PARP1, the protein that was long regarded as a primary nick sensor, has now been established to play a role of the initial sensor of all DNA discontinuities including DSBs [14,[59][60][61]. PARP1 and its homolog PARP2 are multidomain proteins with a flexible architecture, capable of binding DNA in several modes [60,[62][63][64][65][66][67]. Once activated by DNA binding, PARPs catalyse the autoPARylation and PARylation of other targets, including DNA repair and chromatin proteins. ...
CRISPR/Cas9 system is а powerful gene editing tool based on the RNA-guided cleavage of target DNA. The Cas9 activity can be modulated by proteins involved in DNA damage signalling and repair due to their interaction with double- and single-strand breaks (DSB and SSB, respectively) generated by wild-type Cas9 or Cas9 nickases. Here we address the interplay between Streptococcus pyogenes Cas9 and key DNA repair factors, including poly(ADP-ribose) polymerase 1 (SSB/DSB sensor), its closest homolog poly(ADP-ribose) polymerase 2, Ku antigen (DSB sensor), DNA ligase I (SSB sensor), replication protein A (DNA duplex destabilizer), and Y-box binding protein 1 (RNA/DNA binding protein). None of those significantly affected Cas9 activity, while Cas9 efficiently shielded DSBs and SSBs from their sensors. Poly(ADP-ribosyl)ation of Cas9 detected for poly(ADP-ribose) polymerase 2 had no apparent effect on the activity. In cellulo , Cas9-dependent gene editing was independent of poly(ADP-ribose) polymerase 1. Thus, Cas9 can be regarded as an enzyme mostly orthogonal to the natural regulation of human systems of DNA break sensing and repair.
... Recent studies demonstrate that the affinity of PARP2 for nucleosomal DNA is higher than for naked DNA (13), and WGR domain is responsible for the binding to nucleosomal DNA (19,20). ...
Poly(ADP-ribose)polymerase 2 (PARP2) is a nuclear protein that acts as a DNA damage sensor; it recruits the repair enzymes to a DNA damage site and facilitates formation of the repair complex. Using single particle Förster resonance energy transfer microscopy and electrophoretic mobility shift assay (EMSA) we demonstrated that PARP2 forms complexes with a nucleosome containing different number of PARP2 molecules without altering conformation of nucleosomal DNA both in the presence and in the absence of Mg ²⁺ or Ca ²⁺ ions. In contrast, Zn ²⁺ ions directly interact with PARP2 inducing a local alteration of the secondary structure of the protein and PARP2-mediated, reversible structural reorganization of nucleosomal DNA. AutoPARylation activity of PARP2 is enhanced by Mg ²⁺ ions and modulated by Zn ²⁺ ions: suppressed or enhanced depending on the occupancy of two functionally different Zn ²⁺ binding sites. The data suggest that Zn ²⁺ /PARP2-induced nucleosome reorganization and transient changes in the concentration of the cations could modulate PARP2 activity and the DNA damage response.
Significance Statement
PARP2 recognizes and binds DNA damage sites, recruits the repair enzymes to these sites and facilitates formation of the repair complex. Zn ²⁺ -induced structural reorganization of nucleosomal DNA in the complex with PARP2, which is reported in the paper, could modulate the DNA damage response. The obtained data indicate the existence of specific binding sites of Mg ²⁺ and Zn ²⁺ ions in and/or near the catalytic domain of PARP2, which modulate strongly, differently and ion-specifically PARylation activity of PARP2, which is important for maintaining genome stability, adaptation of cells to stress, regulation of gene expression and antioxidant defense.
... Similar to PRMT5 and APE1, the downregulation of SUPT16H significantly sensitized a breast cancer cell line MCF7 to IR [42]. PARP2, which has recently been shown to bind to DSBs with histone parylation factor 1 (HPF1), presumably to facilitate DSB rejoining [43], is also in the region; its expression is highly correlated with that of PRMT5 and APE1. Other genes in the cluster have also been reported for their involvement in DNA damage responses. ...
Genome instability in cancer cells causes not only point mutations but also structural variations of the genome, including copy number variations (CNVs). It has recently been proposed that CNVs arise in cancer to adapt to a given microenvironment to survive. However, how CNV influences cellular resistance against ionizing radiation remains unknown. PRMT5 (protein arginine methyltransferase 5) and APE1 (apurinic/apyrimidinic endonuclease 1), which enhance repair of DNA double-strand breaks and oxidative DNA damage, are closely localized in the chromosome 14 of the human genome. In this study, the genomics data for the PRMT5 and APE1 genes, including their expression, CNVs, and clinical outcomes, were analyzed using TCGA’s data set for oral squamous cell carcinoma patients. The two genes were found to share almost identical CNV values among cancer tissues from oral squamous cell carcinoma (OSCC) patients. Levels of expression of PRMT5 and APE1 in OSCC tissues are highly correlated in cancer but not in normal tissues, suggesting that regulation of PRMT5 and APE1 were overridden by the extent of CNV in the PRMT5-APE1 genome region. High expression levels of PRMT5 and APE1 were both associated with poor survival outcomes after radiation therapy. Simultaneous down-regulation of PRMT5 and APE1 synergistically hampered DNA double-strand break repair and sensitized OSCC cell lines to X-ray irradiation in vitro and in vivo. These results suggest that the extent of CNV in a particular genome region significantly influence the radiation resistance of cancer cells. Profiling CNV in the PRMT5-APE1 genome region may help us to understand the mechanism of the acquired radioresistance of tumor cells, and raises the possibility that simultaneous inhibition of PRMT5 and APE1 may increase the efficacy of radiation therapy.
... Thus, the amino-acid residues in the WGR domain of PARP2 play a key role in the interaction of full-sized PARP2 with DNA, and the N129 residue is a key element in the transmission of the activation signal from WGR to CAT; in this case, two PARP2 molecules form a bridge between two DNA oligomers [51]. Two papers have recently been published concerning the study of the structure of the PARP2 complex with nucleosomes [52,53], which is formed primarily with the participation of the WGR domain. S. Bilokapic et al. [53] have found in the crystallized HPF1-PARP2-nucleosome complex a bridge between two nucleosomes, which connects the free ends of nucleosomal DNA. ...
... Two papers have recently been published concerning the study of the structure of the PARP2 complex with nucleosomes [52,53], which is formed primarily with the participation of the WGR domain. S. Bilokapic et al. [53] have found in the crystallized HPF1-PARP2-nucleosome complex a bridge between two nucleosomes, which connects the free ends of nucleosomal DNA. This bridge is formed by two PARP2-HPF1 complexes; the PARP2 WGR domains connect the DNA break, thereby holding two nucleosomes together. ...
... The key residues of PARP2 WGR are described, which include the important R140 residue located in the WGR signal loop. Mutation of this residue prevents the binding of nucleosomes and the formation of bridges, which explains the rapid dissociation of R140-mutant PARP2 from chromatin in vivo [53]. ...
... PARP1, the protein that was long regarded as a primary nick sensor, has now been established to play a role of the initial sensor of all DNA discontinuities including DSBs [14,[59][60]. PARP1 and its homolog PARP2 are multidomain proteins with a flexible architecture, capable of binding DNA in several modes [60,[62][63][64][65][66][67]. Once activated by DNA binding, PARPs catalyse the auto-PARylation and PARylation of other targets, including DNA repair and chromatin proteins. ...
CRISPR/Cas9 system is а powerful gene editing tool based on the RNA-guided cleavage of target DNA. The Cas9 activity can be modulated by proteins involved in DNA damage signalling and repair due to their interaction with double- and single-strand breaks (DSB and SSB, respectively) generated by wild-type Cas9 or Cas9 nickases. Here we address the interplay between Streptococcus pyogenes Cas9 and key DNA repair factors, including poly(ADP-ribose) polymerase 1 (SSB/DSB sensor), its closest homolog poly(ADP-ribose) polymerase 2, Ku antigen (DSB sensor), DNA ligase I (SSB sensor), replication protein A (DNA duplex destabilizer), and Y-box binding protein 1 (RNA/DNA binding protein). None of those significantly affected Cas9 activity, while Cas9 efficiently shielded DSBs and SSBs from their sensors. Poly(ADP-ribosyl)ation of Cas9 detected for poly(ADP-ribose) polymerase 2 had no apparent effect on the activity. In cellulo , Cas9-dependent gene editing was independent of poly(ADP-ribose) polymerase 1. Thus, Cas9 can be regarded as an enzyme mostly orthogonal to the natural regulation of human systems of DNA break sensing and repair.
... Although the exact context in which HPF1 reshapes the catalytic site of PARP-1 in response to DNA damage is still poorly understood, it switches PARP-1 ADP-ribosylation from carboxylate esters linkages (ASP / GLU residues) to acetal linkages (SER, THR, TYR) ( 161 ). Accumulating evidence suggests that acetal PARylation coordinates DNA repair with cell cycle progression to maintain genome stability (162)(163)(164)(165)(166)(167)(168)(169). Notably, HPF1-dependent histone ADP-ribosylation by PARP-1 contributes to DNA damage-induced chromatin relaxation and promotes the recruitment of repair factors at sites of DNA damage ( 163 ). ...
Poly(ADP-ribosylation) (PARylation) by poly(ADP-ribose) polymerases (PARPs) is a highly regulated process that consists of the covalent addition of polymers of ADP-ribose (PAR) through post-translational modifications of substrate proteins or non-covalent interactions with PAR via PAR binding domains and motifs, thereby reprogramming their functions. This modification is particularly known for its central role in the maintenance of genomic stability. However, how genomic integrity is controlled by an intricate interplay of covalent PARylation and non-covalent PAR binding remains largely unknown. Of importance, PARylation has caught recent attention for providing a mechanistic basis of synthetic lethality involving PARP inhibitors (PARPi), most notably in homologous recombination (HR)-deficient breast and ovarian tumors. The molecular mechanisms responsible for the anti-cancer effect of PARPi are thought to implicate both catalytic inhibition and trapping of PARP enzymes on DNA. However, the relative contribution of each on tumor-specific cytotoxicity is still unclear. It is paramount to understand these PAR-dependent mechanisms, given that resistance to PARPi is a challenge in the clinic. Deciphering the complex interplay between covalent PARylation and non-covalent PAR binding and defining how PARP trapping and non-trapping events contribute to PARPi anti-tumour activity is essential for developing improved therapeutic strategies. With this perspective, we review the current understanding of PARylation biology in the context of the DNA damage response (DDR) and the mechanisms underlying PARPi activity and resistance.
