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1.
Vilchez Larrea SC Alonso GD Schlesinger M Torres HN Flawiá MM Fernández Villamil SH 《International journal for parasitology》2011,41(3-4):405-416
Poly(ADP-ribosyl)ation is a post-translational modification of proteins. Poly(ADP-ribose) polymerase (PARP) and poly(ADP-ribose) glycohydrolase (PARG) are the enzymes responsible for poly(ADP-ribose) (PAR) polymer metabolism and are present in most higher eukaryotes. The best understood role of PARP is the maintenance of genomic integrity either via promotion of DNA repair at low levels of genotoxic stress or via promotion of cell death at higher levels of damage. The unicellular eukaryote Trypanosoma cruzi, as opposed to humans and other organisms, has only one PARP (TcPARP) and one PARG (TcPARG). In the present study we show that under different DNA-damaging agents (H(2)O(2) or UV-C radiation) TcPARP is activated and translocated from the cytosol to the nucleus, while TcPARG always shows a nuclear localisation. Parasites in the presence of PARP or PARG inhibitors, as well as parasites over-expressing either TcPARP or TcPARG, suggested that PAR metabolism could be involved in different phases of cell growth, even in the absence of DNA damage. We also believe that we provide the first reported evidence that different proteins could be poly(ADP-ribosyl)ated in response to different stimuli, leading to different cell death pathways. 相似文献
2.
《Journal of molecular biology》2019,431(15):2655-2673
Nuclear poly(ADP-ribose) polymerases 1 and 2 (PARP1 and PARP2) catalyze the synthesis of poly(ADP-ribose) (PAR) and use NAD+ as a substrate for the polymer synthesis. Both PARP1 and PARP2 are involved in DNA damage response pathways and function as sensors of DNA breaks, including temporary single-strand breaks formed during DNA repair. Consistently, with a role in DNA repair, PARP activation requires its binding to a damaged DNA site, which initiates PAR synthesis. Here we use atomic force microscopy to characterize at the single-molecule level the interaction of PARP1 and PARP2 with long DNA substrates containing a single damage site and representing intermediates of the short-patch base excision repair (BER) pathway. We demonstrated that PARP1 has higher affinity for early intermediates of BER than PARP2, whereas both PARPs efficiently interact with the nick and may contribute to regulation of the final ligation step. The binding of a DNA repair intermediate by PARPs involved a PARP monomer or dimer depending on the type of DNA damage. PARP dimerization influences the affinity of these proteins to DNA and affects their enzymatic activity: the dimeric form is more effective in PAR synthesis in the case of PARP2 but is less effective in the case of PARP1. PARP2 suppresses PAR synthesis catalyzed by PARP1 after single-strand breaks formation. Our study suggests that the functions of PARP1 and PARP2 overlap in BER after a site cleavage and provides evidence for a role of PARP2 in the regulation of PARP1 activity. 相似文献
3.
Gao H Coyle DL Meyer-Ficca ML Meyer RG Jacobson EL Wang ZQ Jacobson MK 《Experimental cell research》2007,313(5):984-996
Genotoxic stress activates nuclear poly(ADP-ribose) (PAR) metabolism leading to PAR synthesis catalyzed by DNA damage activated poly(ADP-ribose) polymerases (PARPs) and rapid PAR turnover by action of nuclear poly(ADP-ribose) glycohydrolase (PARG). The involvement of PARP-1 and PARP-2 in responses to DNA damage has been well studied but the involvement of nuclear PARG is less well understood. To gain insights into the function of nuclear PARG in DNA damage responses, we have quantitatively studied PAR metabolism in cells derived from a hypomorphic mutant mouse model in which exons 2 and 3 of the PARG gene have been deleted (PARG-Delta2,3 cells), resulting in a nuclear PARG containing a catalytic domain but lacking the N-terminal region (A domain) of the protein. Following DNA damage induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), we found that the activity of both PARG and PARPs in intact cells is increased in PARG-Delta2,3 cells. The increased PARG activity leads to decreased PARP-1 automodification with resulting increased PARP activity. The degree of PARG activation is greater than PARP, resulting in decreased PAR accumulation. Following MNNG treatment, PARG-Delta2,3 cells show reduced formation of XRCC1 foci, delayed H2AX phosphorylation, decreased DNA break intermediates during repair, and increased cell death. Our results show that a precise coordination of PARPs and PARG activities is important for normal cellular responses to DNA damage and that this coordination is defective in the absence of the PARG A domain. 相似文献
4.
Salomé C. Vilchez Larrea Teemu Haikarainen Mohit Narwal Mariana Schlesinger Harikanth Venkannagari Mirtha M. Flawiá Silvia H. Fernández Villamil Lari Lehti? 《PloS one》2012,7(9)
Poly(ADP-ribosylation) is a post-translational covalent modification of proteins catalyzed by a family of enzymes termed poly(ADP-ribose) polymerases (PARPs). In the human genome, 17 different genes have been identified that encode members of the PARP superfamily. Poly (ADP-ribose) metabolism plays a role in a wide range of biological processes. In Trypanosoma cruzi, PARP enzyme appears to play a role in DNA repair mechanisms and may also be involved in controlling the different phases of cell growth. Here we describe the identification of potent inhibitors for T. cruzi PARP with a fluorescence-based activity assay. The inhibitors were also tested on T. cruzi epimastigotes, showing that they reduced ADP-ribose polymer formation in vivo. Notably, the identified inhibitors are able to reduce the growth rate of T. cruzi epimastigotes. The best inhibitor, Olaparib, is effective at nanomolar concentrations, making it an efficient chemical tool for chacterization of ADP-ribose metabolism in T. cruzi. PARP inhibition also decreases drastically the amount of amastigotes but interestingly has no effect on the amount of trypomastigotes in the cell culture. Knocking down human PARP-1 decreases both the amount of amastigotes and trypomastigotes in cell culture, indicating that the effect would be mainly due to inhibition of human PARP-1. The result suggests that the inhibition of PARP could be a potential way to interfere with T. cruzi infection. 相似文献
5.
Ekaterina A. Maltseva Nadejda I. Rechkunova Maria V. Sukhanova Olga I. Lavrik 《The Journal of biological chemistry》2015,290(36):21811-21820
Poly(ADP-ribosyl)ation is a reversible post-translational modification that plays an essential role in many cellular processes, including regulation of DNA repair. Cellular DNA damage response by the synthesis of poly(ADP-ribose) (PAR) is mediated mainly by poly(ADP-ribose) polymerase 1 (PARP1). The XPC-RAD23B complex is one of the key factors of nucleotide excision repair participating in the primary DNA damage recognition. By using several biochemical approaches, we have analyzed the influence of PARP1 and PAR synthesis on the interaction of XPC-RAD23B with damaged DNA. Free PAR binds to XPC-RAD23B with an affinity that depends on the length of the poly(ADP-ribose) strand and competes with DNA for protein binding. Using 32P-labeled NAD+ and immunoblotting, we also demonstrate that both subunits of the XPC-RAD23B are poly(ADP-ribosyl)ated by PARP1. The efficiency of XPC-RAD23B PARylation depends on DNA structure and increases after UV irradiation of DNA. Therefore, our study clearly shows that XPC-RAD23B is a target of poly(ADP-ribosyl)ation catalyzed by PARP1, which can be regarded as a universal regulator of DNA repair processes. 相似文献
6.
Poly(ADP-ribosyl)ation (PARylation) of proteins is one of the immediate cell responses to DNA damage and is catalyzed by poly(ADP-ribose) polymerases (PARPs). When bound to damaged DNA, some members of the PARP family are activated and use NAD+ as a source of ADP to catalyze synthesis of poly(ADP-ribose) (PAR) covalently attached to a target protein. PAR synthesis is considered as a mechanism that provides a local signal of DNA damage and modulates protein functions in response to genotoxic agents. PARP1 is the best-studied protein of the PARP family and is widely known аs a regulator of repair of damaged bases and single-strand nicks. Data are accumulating that PARP1 is additionally involved in double-strand break repair and nucleotide excision repair. The review summarizes the literature data on the role that PARP1 and PARylation play in DNA repair and particularly in base excision repair; original data obtained in our lab are considered in more detail. 相似文献
7.
Poly(ADP-ribose) polymerase 1 (PARP1), an enzyme activated by DNA strand breaks, synthesizes polymers of poly(ADP-ribose) (PAR) that modify chromatin and other proteins and play a role in DNA repair. Inhibition of PARP1 activity is considered a potentially important strategy in clinical practice, especially to sensitize tumor cells to chemo- and radio-therapy. Here we examined the influence of inhibition of PARP1 on formation of reactive oxygen species (ROS) and on DNA repair in cells exposed to ionizing radiation (IR). K562 (human myelogenous leukaemia) cells were grown and exposed to 4 or 12Gy of ionizing radiation in presence or absence of the PARP inhibitor NU1025 (100μM). Intracellular ROS were assayed using the probe 2,7-dichlorofluorescein with detection by flow cytometry and the rejoining of DNA strand breaks were followed by alkaline single cell gel electrophoresis (comet) assays. In untreated cells a significant increase in PAR formation occurred during the first 5min after IR, followed by a gradual decrease up to 30min. Addition of a PARP inhibitor arrested the production of PAR almost completely and decreased the rate of rejoining of DNA strand breaks significantly; however, 3h after irradiation we observed no difference in the amount of DNA strand breaks between PARP inhibitor-treated and untreated cells. Twelve to 48h after irradiation, an increase of ROS concentration was observed in irradiated cells and ROS levels in PARP inhibitor-treated cells were significantly higher than in cells without inhibitor. Irradiated cells grown in the presence or absence of PARP inhibitor did not differ in the frequencies of apoptotic and necrotic cells or in the activity of caspases at 24, 48 and 72h after irradiation. Poly(ADP-ribosylation) and inhibition of PARP1 appeared to modulate DNA strand break rejoining and influence the concentration of ROS in irradiated cells. 相似文献
8.
Mortusewicz O Fouquerel E Amé JC Leonhardt H Schreiber V 《Nucleic acids research》2011,39(12):5045-5056
Post-translational poly(ADP-ribosyl)ation has diverse essential functions in the cellular response to DNA damage as it contributes to avid DNA damage detection and assembly of the cellular repair machinery but extensive modification eventually also induces cell death. While there are 17 human poly(ADP-ribose) polymerase (PARP) genes, there is only one poly(ADP-ribose) glycohydrolase (PARG) gene encoding several PARG isoforms located in different subcellular compartments. To investigate the recruitment of PARG isoforms to DNA repair sites we locally introduced DNA damage by laser microirradiation. All PARG isoforms were recruited to DNA damage sites except for a mitochondrial localized PARG fragment. Using PARP knock out cells and PARP inhibitors, we showed that PARG recruitment was only partially dependent on PARP-1 and PAR synthesis, indicating a second, PAR-independent recruitment mechanism. We found that PARG interacts with PCNA, mapped a PCNA binding site and showed that binding to PCNA contributes to PARG recruitment to DNA damage sites. This dual recruitment mode of the only nuclear PARG via the versatile loading platform PCNA and by a PAR dependent mechanism likely contributes to the dynamic regulation of this posttranslational modification and ensures the tight control of the switch between efficient DNA repair and cell death. 相似文献
9.
Herpes simplex virus 1 infection triggers multiple changes in the metabolism of host cells, including a dramatic decrease in the levels of NAD(+). In addition to its role as a cofactor in reduction-oxidation reactions, NAD(+) is required for certain posttranslational modifications. Members of the poly(ADP-ribose) polymerase (PARP) family of enzymes are major consumers of NAD(+), which they utilize to form poly(ADP-ribose) (PAR) chains on protein substrates in response to DNA damage. PAR chains can subsequently be removed by the enzyme poly(ADP-ribose) glycohydrolase (PARG). We report here that the HSV-1 infection-induced drop in NAD(+) levels required viral DNA replication, was associated with an increase in protein poly(ADP-ribosyl)ation (PARylation), and was blocked by pharmacological inhibition of PARP-1/PARP-2 (PARP-1/2). Neither virus yield nor the cellular metabolic reprogramming observed during HSV-1 infection was altered by the rescue or further depletion of NAD(+) levels. Expression of the viral protein ICP0, which possesses E3 ubiquitin ligase activity, was both necessary and sufficient for the degradation of the 111-kDa PARG isoform. This work demonstrates that HSV-1 infection results in changes to NAD(+) metabolism by PARP-1/2 and PARG, and as PAR chain accumulation can induce caspase-independent apoptosis, we speculate that the decrease in PARG levels enhances the auto-PARylation-mediated inhibition of PARP, thereby avoiding premature death of the infected cell. 相似文献
10.
《DNA Repair》2017
Poly(ADP-ribosylation) of proteins following DNA damage is well studied and the use of poly(ADP-ribose) polymerase (PARP) inhibitors as therapeutic agents is an exciting prospect for the treatment of many cancers. Poly(ADP-ribose) glycohydrolase (PARG) has endo- and exoglycosidase activities which can cleave glycosidic bonds, rapidly reversing the action of PARP enzymes. Like addition of poly(ADP-ribose) (PAR) by PARP, removal of PAR by PARG is also thought to be required for repair of DNA strand breaks and for continued replication at perturbed forks. Here we use siRNA to show a synthetic lethal relationship between PARG and BRCA1, BRCA2, PALB2, FAM175A (ABRAXAS) and BARD1. In addition, we demonstrate that MCF7 cells depleted of these proteins are sensitive to Gallotannin and a novel and specific PARG inhibitor PDD00017273. We confirm that PARG inhibition increases endogenous DNA damage, stalls replication forks and increases homologous recombination, and propose that it is the lack of homologous recombination (HR) proteins at PARG inhibitor-induced stalled replication forks that induces cell death. Interestingly not all genes that are synthetically lethal with PARP result in sensitivity to PARG inhibitors, suggesting that although there is overlap, the functions of PARP and PARG may not be completely identical. These data together add further evidence to the possibility that single treatment therapy with PARG inhibitors could be used for treatment of certain HR deficient tumours and provide insight into the relationship between PARP, PARG and the processes of DNA repair. 相似文献
11.
Benu Brata Das Shar-yin N. Huang Junko Murai Ishita Rehman Jean-Christophe Amé Souvik Sengupta Subhendu K. Das Papiya Majumdar Hongliang Zhang Denis Biard Hemanta K. Majumder Valérie Schreiber Yves Pommier 《Nucleic acids research》2014,42(7):4435-4449
Poly(ADP-ribose) polymerases (PARP) attach poly(ADP-ribose) (PAR) chains to various proteins including themselves and chromatin. Topoisomerase I (Top1) regulates DNA supercoiling and is the target of camptothecin and indenoisoquinoline anticancer drugs, as it forms Top1 cleavage complexes (Top1cc) that are trapped by the drugs. Endogenous and carcinogenic DNA lesions can also trap Top1cc. Tyrosyl-DNA phosphodiesterase 1 (TDP1), a key repair enzyme for trapped Top1cc, hydrolyzes the phosphodiester bond between the DNA 3′-end and the Top1 tyrosyl moiety. Alternative repair pathways for Top1cc involve endonuclease cleavage. However, it is unknown what determines the choice between TDP1 and the endonuclease repair pathways. Here we show that PARP1 plays a critical role in this process. By generating TDP1 and PARP1 double-knockout lymphoma chicken DT40 cells, we demonstrate that TDP1 and PARP1 are epistatic for the repair of Top1cc. The N-terminal domain of TDP1 directly binds the C-terminal domain of PARP1, and TDP1 is PARylated by PARP1. PARylation stabilizes TDP1 together with SUMOylation of TDP1. TDP1 PARylation enhances its recruitment to DNA damage sites without interfering with TDP1 catalytic activity. TDP1–PARP1 complexes, in turn recruit X-ray repair cross-complementing protein 1 (XRCC1). This work identifies PARP1 as a key component driving the repair of trapped Top1cc by TDP1. 相似文献
12.
Poly(ADP-ribose) polymerase (PARP) is an intracellular enzyme involved in DNA repair and in building poly-ADP-ribose polymers on nuclear proteins using NAD+. While the majority of PARP resides in the nucleus, several studies indicated that PARP may also be located in the cytosol or in the mitochondrial matrix. In this study we found several poly-ADP-ribosylated proteins in isolated rat liver mitochondria following hydrogen peroxide (H2O2) or nitric oxide donor treatment. Protein poly-ADP-ribosylation was more intense in isolated mitochondria than in whole tissue homogenates and it was not associated with increased nuclear PARP activity. We identified five poly-ADP-ribose (PAR) positive mitochondrial bands by protein mass fingerprinting. All of the identified enzymes exhibited decreased activity or decreased levels following oxidative or nitrosative stress. One of the identified proteins is dihydrolipoamide dehydrogenase (DLDH), a component of the alpha-ketoglutarate dehydrogenase (KGDH) complex, which uses NAD+ as a substrate. This raised the possibility that KGDH may have a PARP-like enzymatic activity. The intrinsic PARP activity of KGDH and DLDH was confirmed using a colorimetric PARP assay kit and by the incubation of the recombinant enzymes with H2O2. The KGDH enzyme may, therefore, have a novel function as a PARP-like enzyme, which may play a role in regulating intramitochondrial NAD+ and poly(ADP-ribose) homeostasis, with possible roles in physiology and pathophysiology. 相似文献
13.
DNA excision repair and DNA damage-induced apoptosis are linked to Poly(ADP-ribosyl)ation but have different requirements for p53 总被引:10,自引:0,他引:10
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Beneke R Geisen C Zevnik B Bauch T Müller WU Küpper JH Möröy T 《Molecular and cellular biology》2000,20(18):6695-6703
Poly(ADP-ribose) polymerase (PARP) is a DNA binding zinc finger protein that catalyzes the transfer of ADP-ribose residues from NAD(+) to itself and different chromatin constituents, forming branched ADP-ribose polymers. The enzymatic activity of PARP is induced upon DNA damage and the PARP protein is cleaved during apoptosis, which suggested a role of PARP in DNA repair and DNA damage-induced cell death. We have generated transgenic mice that lack PARP activity in thymocytes owing to the targeted expression of a dominant negative form of PARP. In the presence of single-strand DNA breaks, the absence of PARP activity correlated with a strongly increased rate of apoptosis compared to cells with intact PARP activity. We found that blockage of PARP activity leads to a drastic increase of p53 expression and activity after DNA damage and correlates with an accelerated onset of Bax expression. DNA repair is almost completely blocked in PARP-deficient thymocytes regardless of p53 status. We found the same increased susceptibility to apoptosis in PARP null mice, a similar inhibition of DNA repair kinetics, and the same upregulation of p53 in response to DNA damage. Thus, based on two different experimental in vivo models, we identify a direct, p53-independent, functional connection between poly(ADP-ribosyl)ation and the DNA excision repair machinery. Furthermore, we propose a p53-dependent link between PARP activity and DNA damage-induced cell death. 相似文献
14.
Meyer-Ficca ML Lonchar JD Ihara M Meistrich ML Austin CA Meyer RG 《Biology of reproduction》2011,84(5):900-909
To achieve the specialized nuclear structure in sperm necessary for fertilization, dramatic chromatin reorganization steps in developing spermatids are required where histones are largely replaced first by transition proteins and then by protamines. This entails the transient formation of DNA strand breaks to allow for, first, DNA relaxation and then chromatin compaction. However, the nature and origin of these breaks are not well understood. We previously reported that these DNA strand breaks trigger the activation of poly(ADP-ribose) (PAR) polymerases PARP1 and PARP2 and that interference with PARP activation causes poor chromatin integrity with abnormal retention of histones in mature sperm and impaired embryonic survival. Here we show that the activity of topoisomerase II beta (TOP2B), an enzyme involved in DNA strand break formation in elongating spermatids, is strongly inhibited by the activity of PARP1 and PARP2 in vitro, and this is in turn counteracted by the PAR-degrading activity of PAR glycohydrolase. Moreover, genetic and pharmacological PARP inhibition both lead to increased TOP2B activity in murine spermatids in vivo as measured by covalent binding of TOP2B to the DNA. In summary, the available data suggest a functional relationship between the DNA strand break-generating activity of TOP2B and the DNA strand break-dependent activation of PARP enzymes that in turn inhibit TOP2B. Because PARP activity also facilitates histone H1 linker removal and local chromatin decondensation, cycles of PAR formation and degradation may be necessary to coordinate TOP2B-dependent DNA relaxation with histone-to-protamine exchange necessary for spermatid chromatin remodeling. 相似文献
15.
Nagayama T Simon RP Chen D Henshall DC Pei W Stetler RA Chen J 《Journal of neurochemistry》2000,74(4):1636-1645
We have investigated the role of poly(ADP-ribose) polymerase (PARP) activation in rat brain in a model of sublethal transient global ischemia. Adult male rats were subjected to 15 min of ischemia with brain temperature reduced to 34 degrees C, followed by 1, 2, 4, 8, 16, 24, and 72 h of reperfusion. PARP mRNA expression was examined in the hippocampus using quantitative RT-PCR, northern blot analysis, and in situ hybridization. Protein expression was assessed using western blot analysis. PARP enzymatic activity was investigated by measuring nuclear [3H]NAD incorporation. The presence of poly(ADP-ribose) polymers was assessed immunocytochemically. Although PARP mRNA and protein expressions were not altered after ischemia, enzymatic activity was increased 4.37-fold at 1 h (p < 0.05 vs. sham) and 1.73-fold (p < 0.05 vs. sham) at 24 h of reperfusion. Immunostaining demonstrated the presence of poly(ADP-ribose) polymers in CA1 neurons. Cellular NAD+ levels were not significantly altered at any time point. Furthermore, systemic administration of 3-aminobenzamide (30 mg/kg), a PARP inhibitor, prevented the increase in PARP activity at 1 and 24 h of reperfusion, significantly decreased the number of surviving neurons in the hippocampal CA1 region 72 h after ischemia (p < 0.01 vs. sham), and increased DNA single-strand breaks assessed as DNA polymerase I-mediated biotin-dATP nick-translation (PANT)-positive cells (p < 0.01 vs. sham). Furthermore, using an in vitro DNA repair assay, 3-aminobenzamide (30 mg/kg) was shown to block DNA base excision repair activity. These data suggest that the activation of PARP, without subsequent NAD+ depletion, following mild transient ischemia may be neuroprotective in the brain. 相似文献
16.
Niere M Mashimo M Agledal L Dölle C Kasamatsu A Kato J Moss J Ziegler M 《The Journal of biological chemistry》2012,287(20):16088-16102
17.
Brenee S. King Karen L. Cooper Ke Jian Liu Laurie G. Hudson 《The Journal of biological chemistry》2012,287(47):39824-39833
Exposure to ultraviolet radiation (UVR) promotes the formation of UVR-induced, DNA helix distorting photolesions such as (6-4) pyrimidine-pyrimidone photoproducts and cyclobutane pyrimidine dimers. Effective repair of such lesions by the nucleotide excision repair (NER) pathway is required to prevent DNA mutations and chromosome aberrations. Poly(ADP-ribose) polymerase-1 (PARP-1) is a zinc finger protein with well documented involvement in base excision repair. PARP-1 is activated in response to DNA damage and catalyzes the formation of poly(ADP-ribose) subunits that assist in the assembly of DNA repair proteins at sites of damage. In this study, we present evidence for PARP-1 contributions to NER, extending the knowledge of PARP-1 function in DNA repair beyond the established role in base excision repair. Silencing the PARP-1 protein or inhibiting PARP activity leads to retention of UVR-induced photolesions. PARP activation following UVR exposure promotes association between PARP-1 and XPA, a central protein in NER. Administration of PARP inhibitors confirms that poly(ADP-ribose) facilitates PARP-1 association with XPA in whole cell extracts, in isolated chromatin complexes, and in vitro. Furthermore, inhibition of PARP activity decreases UVR-stimulated XPA chromatin association, illustrating that these relationships occur in a meaningful context for NER. These results provide a mechanistic link for PARP activity in the repair of UVR-induced photoproducts. 相似文献
18.
19.
《Biochimica et Biophysica Acta - Proteins and Proteomics》2019,1867(3):297-305
Base excision repair (BER) involves many enzymes acting in a coordinated fashion at the most common types of DNA damage. The coordination is facilitated by interactions between the enzymes and accessory proteins, X-ray repair cross-complementing protein 1 (XRCC1) and poly(ADP-ribose) polymerase 1 (PARP1). Here we use dynamic light scattering (DLS) technique to determine the hydrodynamic sizes of several BER enzymes and proteins, DNA polymerase β (Polβ), apurinic/apyrimidinic endonuclease 1 (APE1), tyrosyl-DNA phosphodiesterase 1 (TDP1), XRCC1 and PARP1, present alone or in the equimolar mixtures with each other. From the DLS data combined with glutaraldehyde cross-linking experiments and previous quantitative binding data the oligomeric states of BER proteins and their complexes are estimated. All the proteins have been proposed to form homodimers upon their self-association. The most probable oligomerization state of the binary complexes formed by PARP1 with various proteins is a heterotetramer. The oligomerization state of the binary complexes formed by XRCC1 varies from heterodimer to heterotetramer, depending on the partner. The DLS technique is applied for the first time to measure the hydrodynamic sizes of PARP1 molecules covalently bound with poly(ADP-ribose) (PAR) synthesized upon the automodification reaction. PARP1 has been detected to form huge conglomerates stabilized by Mg2+ coordinated bonds with PAR polymers. 相似文献
20.
Matthias Altmeyer Simon Messner Paul O. Hassa Monika Fey Michael O. Hottiger 《Nucleic acids research》2009,37(11):3723-3738
Poly(ADP-ribose) polymerase 1 (PARP1) synthesizes poly(ADP-ribose) (PAR) using nicotinamide adenine dinucleotide (NAD) as a substrate. Despite intensive research on the cellular functions of PARP1, the molecular mechanism of PAR formation has not been comprehensively understood. In this study, we elucidate the molecular mechanisms of poly(ADP-ribosyl)ation and identify PAR acceptor sites. Generation of different chimera proteins revealed that the amino-terminal domains of PARP1, PARP2 and PARP3 cooperate tightly with their corresponding catalytic domains. The DNA-dependent interaction between the amino-terminal DNA-binding domain and the catalytic domain of PARP1 increased Vmax and decreased the Km for NAD. Furthermore, we show that glutamic acid residues in the auto-modification domain of PARP1 are not required for PAR formation. Instead, we identify individual lysine residues as acceptor sites for ADP-ribosylation. Together, our findings provide novel mechanistic insights into PAR synthesis with significant relevance for the different biological functions of PARP family members. 相似文献