Physical and functional interaction between DNA ligase IIIalpha and poly(ADP-Ribose) polymerase 1 in DNA single-strand break repair

The repair of DNA single-strand breaks in mammalian cells is mediated by poly(ADP-ribose) polymerase 1 (PARP-1), DNA ligase IIIalpha, and XRCC1. Since these proteins are not found in lower eukaryotes, this DNA repair pathway plays a unique role in maintaining genome stability in more complex organisms. XRCC1 not only forms a stable complex with DNA ligase IIIalpha but also interacts with several other DNA repair factors. Here we have used affinity chromatography to identify proteins that associate with DNA ligase III. PARP-1 binds directly to an N-terminal region of DNA ligase III immediately adjacent to its zinc finger. In further studies, we have shown that DNA ligase III also binds directly to poly(ADP-ribose) and preferentially associates with poly(ADP-ribosyl)ated PARP-1 in vitro and in vivo. Our biochemical studies have revealed that the zinc finger of DNA ligase III increases DNA joining in the presence of either poly(ADP-ribosyl)ated PARP-1 or poly(ADP-ribose). This provides a mechanism for the recruitment of the DNA ligase IIIalpha-XRCC1 complex to in vivo DNA single-strand breaks and suggests that the zinc finger of DNA ligase III enables this complex and associated repair factors to locate the strand break in the presence of the negatively charged poly(ADP-ribose) polymer.     

二磷酸腺苷核糖多聚酶的研究进展

二磷酸腺苷核糖多聚酶[Poly（ADP-Ribose）Polymerase,PAPe]是一类具有重要生理功能的蛋白酶。PARP能催化二磷酸腺苷核糖多聚化反应。二磷酸腺苷核糖多聚化对DNA修复和基因组稳定性起着重要作用。但PARP的过激活与许多疾病的病理机制有关。介绍了PARP的结构和功能,PARP家族的同族体以及PARP在一些疾病病理机制中的作用。     

Poly(ADP-Ribose) polymerase inhibition improves endothelial dysfunction induced by hypochlorite

Reactive oxygen species, such as myeloperoxidase-derived hypochlorite, induce oxidative stress and DNA injury. The subsequent activation of the DNA-damage-poly(ADP-ribose) polymerase (PARP) pathway has been implicated in the pathogenesis of various diseases, including ischemia-reperfusion injury, circulatory shock, diabetic complications, and atherosclerosis. We investigated the effect of PARP inhibition on the impaired endothelium-dependent vasorelaxation induced by hypochlorite. In organ bath experiments for isometric tension, we investigated the endothelium-dependent and endothelium-independent vasorelaxation of isolated rat aortic rings using cumulative concentrations of acetylcholine and sodium nitro-prusside. Endothelial dysfunction was induced by exposing rings to hypochlorite (100-400 microM). In the treatment group, rings were preincubated with the PARP inhibitor INO-1001. DNA strand breaks were assessed by the TUNEL method. Immunohistochemistry was performed for 4-hydroxynonenal (a marker of lipid peroxidation), nitrotyrosine (a marker of nitrosative stress), and poly(ADP-ribose) (an enzymatic product of PARP). Exposure to hypochlorite resulted in a dose-dependent impairment of endothelium-dependent vasorelaxation of aortic rings, which was significantly improved by PARP inhibition, whereas the endothelium-independent vasorelaxation remained unaffected. In the hypochlorite groups we found increased DNA breakage, lipidperoxidation, and enhanced nitrotyrosine formation. The hypochloride-induced activation of PARP was prevented by INO-1001. Our results demonstrate that PARP activation contributes to the pathogenesis of hypochlorite-induced endothelial dysfunction, which can be prevented by PARP inhibitors.     

The Use of Biotinylated Poly(ADP-Ribose) for Studies on Poly(ADP-Ribose)-Protein Interaction

Poly(ADP-ribose) is routinely detected by the use of radioactive polymers formed from labeled substrates. In this report a simple and time-saving method for the biotinylation and the detection of poly(ADP-ribose) on blots is described. The polymer modified by light-induced reaction with photobiotin was colorimetrically detected and quantified, using streptavidine-alkaline phosphatase conjugates. The separation of poly(ADP-ribose) chains on polyacrylamide gels was not affected by the biotinylation of the polymers. When biotinylated poly(ADP-ribose) was used to detect the poly(ADP-ribose) binding capability of proteins in ligand blots, the results were comparable to those obtained with poly([³²P]ADP-ribose). Experiments with histones and rat liver nuclear proteins demonstrate that in studies on poly(ADP-ribose)-protein interaction, this method is applicable to the detection of poly(ADP-ribose) binding proteins.     

Differential and Concordant Roles for Poly(ADP-Ribose) Polymerase 1 and Poly(ADP-Ribose) in Regulating WRN and RECQL5 Activities

Poly(ADP-ribose) (PAR) polymerase 1 (PARP1) catalyzes the poly(ADP-ribosyl)ation (PARylation) of proteins, a posttranslational modification which forms the nucleic acid-like polymer PAR. PARP1 and PAR are integral players in the early DNA damage response, since PARylation orchestrates the recruitment of repair proteins to sites of damage. Human RecQ helicases are DNA unwinding proteins that are critical responders to DNA damage, but how their recruitment and activities are regulated by PARPs and PAR is poorly understood. Here we report that all human RecQ helicases interact with PAR noncovalently. Furthermore, we define the effects that PARP1, PARylated PARP1, and PAR have on RECQL5 and WRN, using both in vitro and in vivo assays. We show that PARylation is involved in the recruitment of RECQL5 and WRN to laser-induced DNA damage and that RECQL5 and WRN have differential responses to PARylated PARP1 and PAR. Furthermore, we show that the loss of RECQL5 or WRN resulted in increased sensitivity to PARP inhibition. In conclusion, our results demonstrate that PARP1 and PAR actively, and in some instances differentially, regulate the activities and cellular localization of RECQL5 and WRN, suggesting that PARylation acts as a fine-tuning mechanism to coordinate their functions in time and space during the genotoxic stress response.     

Role of YB-1 in Regulation of Poly(ADP-Ribosylation) Catalyzed by Poly(ADP-Ribose) Polymerases

Biochemistry (Moscow) - Poly(ADP-ribosyl)ation is a post-translational modification of proteins that performs an essential regulatory function in the cellular response to DNA damage. The key enzyme...     

p21CDKN1A Regulates the Binding of Poly(ADP-Ribose) Polymerase-1 to DNA Repair Intermediates

The cell cycle inhibitor p21^CDKN1A was previously found to interact directly with DNA nick-sensor poly(ADP-ribose) polymerase-1 (PARP-1) and to promote base excision repair (BER). However, the molecular mechanism responsible for this BER-related association of p21 with PARP-1 remains to be clarified. In this study we investigate the capability of p21 to influence PARP-1 binding to DNA repair intermediates in a reconstituted BER system in vitro. Using model photoreactive BER substrates containing single-strand breaks, we found that full-length recombinant GST-tagged p21 but not a C-terminal domain truncated form of p21 was able to stimulate the PARP-1 binding to BER intermediates with no significant influence on the catalytic activity of PARP-1. In addition, we investigate whether the activation of PARP-1 through poly(ADP-ribose) (PAR) synthesis, is required for its interaction with p21. We have found that in human fibroblasts and in HeLa cells treated with the DNA alkylating agent N-methyl-N''-nitro-N-nitrosoguanidine (MNNG), the interaction of p21 with PARP-1 was greatly dependent on PAR synthesis. In fact, an anti-PAR antibody was able to co-immunoprecipitate p21 and PARP-1 from extracts of MNNG-treated cells, while blocking PAR synthesis with the PARP-1 inhibitor Olaparib, drastically reduced the amount of p21 co-immunoprecipitated by a PARP-1 antibody. Our results provide the first evidence that p21 can stimulate the binding of PARP-1 to DNA repair intermediates, and that this cooperation requires PAR synthesis.     

Increased DNA Damage in Progression Of COPD: A Response By Poly(ADP-Ribose) Polymerase-1

Chronic oxidative stress (OS), a major mechanism of chronic obstructive pulmonary disease (COPD), may cause significant damage to DNA. Poly(ADP-ribose) polymerase (PARP)-1 is rapidly activated by OS-induced DNA lesions. However, the degree of DNA damage along with the evolution of COPD is unclear. In peripheral blood mononuclear cells of non-smoking individuals, non-obstructive smokers, patients with COPD of all stages and those with COPD exacerbation, we evaluated DNA damage, PARP activity and PARP-1 mRNA expression using Comet Assay IV, biotinylated-NAD incorporation assay and qRT-PCR, respectively and subjected results to ordinal logistic regression modelling. Adjusted for demographics, smoking-related parameters and lung function, novel comet parameters, tail length/cell length ratio and tail migration/cell length ratio, showed the greatest increase along the study groups corresponding to the evolution of COPD [odds ratio (OR) 7.88, 95% CI 4.26–14.57; p<0.001 and OR 3.91, 95% CI 2.69–5.66; p<0.001, respectively]. Analogously, PARP activity increased significantly over the groups (OR = 1.01; 95%; p<0.001). An antioxidant tetrapeptide UPF17 significantly reduced the PARP-1 mRNA expression in COPD, compared to that in non-obstructive individuals (p = 0.040). Tail length/cell length and tail migration/cell length ratios provide novel progression-sensitive tools for assessment of DNA damage. However, it remains to be elucidated whether inhibition of an elevated PARP-1 activity has a safe enough potential to break the vicious cycle of the development and progression of COPD.     

RNA interference directed against Poly(ADP-Ribose) polymerase 1 efficiently suppresses human immunodeficiency virus type 1 replication in human cells

We established small interfering RNA (siRNA) directed against poly(ADP-ribose) polymerase 1 (PARP-1) that effectively reduces the expression of PARP-1 in two human cell lines. Established siRNA against PARP-1 significantly suppressed human immunodeficiency virus type 1 (HIV-1) replication, as well as the activation of the integrated HIV-1 long terminal repeat promoter. These results indicate that PARP-1 is required for efficient HIV-1 replication in human cells. We propose that PARP-1 may serve as a cellular target for RNA interference-mediated gene silencing to inhibit HIV-1 replication.     

Poly(ADP-Ribose) Synthetase Activation: An Early Indicator of Neurotoxic DNA Damage

Abstract: DNA damage activates a nuclear enzyme poly(ADP-ribose) synthetase (PARS) that facilitates DNA repair by adding multiple ADP-ribose groups to nuclear proteins such as histones and PARS itself. N -Methyl- d -aspartate neurotoxicity may involve DNA damage excessively activating PARS to deplete its substrate NAD, as PARS inhibitors prevent this toxicity. We now show that PARS is rapidly and markedly activated in PC12 cells following treatment with neurotoxic agents, including the amyloid β-protein, hydrogen peroxide, N -methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), and its active metabolite N -methyl-4-phenylpyridine (MPP⁺). With MPP⁺, PARS activity is increased fivefold in 1 h and 20-fold by 3 h. By contrast, direct measurement of DNA damage by the terminal-deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling assay shows no significant increase by 3 h and less than fourfold by 24 h. These findings indicate that PARS activity can provide a simple, sensitive, and early index of DNA damage following neurotoxic insults.     

Two-color fluorescence detection of Poly (ADP-Ribose) Polymerase-1 (PARP-1) cleavage and DNA strand breaks in etoposide-induced apoptotic cells

During apoptosis, the nuclear enzyme Poly(ADP-Ribose) Polymerase-1 (PARP-1) catalyzes the rapid and transient synthesis of poly(ADP-ribose) from NAD+ and becomes inactive when cleaved by caspases. The regulation of these two opposite roles of PARP-1 is still unknown. We have recently investigated PARP-1 activation/degradation in Hep-2 cells driven to apoptosis by actinomycin D. In the present work, we have extended our analysis to the effect of the DNA damaging agent etoposide, and paid attention to the relationship between PARP-1 cleavage and DNA fragmentation. An original fluorescent procedure was developed to simultaneously identify in situ the p89 proteolytic fragment of PARP-1 (by immunolabeling) and DNA degradation (by the TUNEL assay). The presence of p89 was observed both in cells with advanced signs of apoptosis (where the PARP-1 fragment is extruded from the nucleus into the cytoplasm) and in TUNEL-negative cells, with only incipient signs of chromatin condensation; this evidence indicates that PARP-1 degradation in etoposide-treated apoptotic cells may precede DNA cleavage.     

DNA polymerase beta -mediated long patch base excision repair. Poly(ADP-ribose)polymerase-1 stimulates strand displacement DNA synthesis.

Recently, photoaffinity labeling experiments with mouse cell extracts suggested that PARP-1 functions as a surveillance protein for a stalled BER intermediate. To further understand the role of PARP-1 in BER, we examined the DNA synthesis and flap excision steps in long patch BER using a reconstituted system containing a 34-base pair BER substrate and five purified human enzymes: uracil-DNA glycosylase, apurinic/apyrimidinic endonuclease, DNA polymerase beta, flap endonuclease-1 (FEN-1), and PARP-1. PARP-1 stimulates strand displacement DNA synthesis by DNA polymerase beta in this system; this stimulation is dependent on the presence of FEN-1. PARP-1 and FEN-1, therefore, cooperate to activate long patch BER. The results are discussed in the context of a model for BER sub-pathway choice, illustrating a dual role for PARP-1 as a surveillance protein for a stalled BER intermediate and an activating factor for long patch BER DNA synthesis.     

Poly(ADP-ribose) polymerase forms loops with DNA

The interaction between highly purified poly(ADP-ribose) polymerase from calf thymus and different topological forms of pBR322 DNA has been studied by gel retardation electrophoresis and electron microscopy. We show that: (i) in the absence of nicks on DNA the enzyme has a marked affinity for supercoiled (form I) DNA, (ii) in the presence of single stranded breaks poly(ADP-ribose) polymerase preferentially binds to form II, (iii) in all cases enzyme molecules are frequently located at DNA intersections, (iv) a cooperative binding of the enzyme on DNA occurs.     

XRCC1 Is Specifically Associated with Poly(ADP-Ribose) Polymerase and Negatively Regulates Its Activity following DNA Damage

Poly(ADP-ribose) polymerase (PARP; EC 2.4.2.30) is a zinc-finger DNA-binding protein that detects and signals DNA strand breaks generated directly or indirectly by genotoxic agents. In response to these breaks, the immediate poly(ADP-ribosyl)ation of nuclear proteins involved in chromatin architecture and DNA metabolism converts DNA damage into intracellular signals that can activate DNA repair programs or cell death options. To have greater insight into the physiological function of this enzyme, we have used the two-hybrid system to find genes encoding proteins putatively interacting with PARP. We have identified a physical association between PARP and the base excision repair (BER) protein XRCC1 (X-ray repair cross-complementing 1) in the Saccharomyces cerevisiae system, which was further confirmed to exist in mammalian cells. XRCC1 interacts with PARP by its central region (amino acids 301 to 402), which contains a BRCT (BRCA1 C terminus) module, a widespread motif in DNA repair and DNA damage-responsive cell cycle checkpoint proteins. Overexpression of XRCC1 in Cos-7 or HeLa cells dramatically decreases PARP activity in vivo, reinforcing the potential protective function of PARP at DNA breaks. Given that XRCC1 is also associated with DNA ligase III via a second BRCT module and with DNA polymerase β, our results provide strong evidence that PARP is a member of a BER multiprotein complex involved in the detection of DNA interruptions and possibly in the recruitment of XRCC1 and its partners for efficient processing of these breaks in a coordinated manner. The modular organizations of these interactors, associated with small conserved domains, may contribute to increasing the efficiency of the overall pathway.The genomic integrity of cells is controlled by a network of protein factors that assess the status of the genome and either cause progression of proliferation or induce a halt in the cell cycle. In eukaryotes, DNA strand breaks, introduced either directly by ionizing radiation or indirectly following enzymatic incision of a DNA lesion, trigger the synthesis of poly(ADP-ribose) by the enzyme poly(ADP-ribose) polymerase (PARP) (1, 13, 39). At the site of breakage, PARP catalyzes the transfer of the ADP-ribose moiety from its substrate, NAD⁺, to a limited number of protein acceptors involved in chromatin architecture and DNA metabolism, including the enzyme itself. These modified proteins, which carry long chains of negatively charged ADP-ribose polymers, lose their affinity for DNA and are thus inactivated. The short half-life of the polymer is attributed to the high activity of poly(ADP-ribose) glycohydrolase, which cleaves the ribose-ribose bond (28, 30). Therefore, poly(ADP-ribosylation) is an immediate but transient postranslational modification of nuclear proteins, induced by DNA-damaging agents.The physiological role of PARP has been much debated in the last decade, but recent molecular and genetic approaches, including expression of either a dominant-negative mutant (26, 36, 44) or antisense oligonucleotides (14), have clearly implicated PARP in the base excision repair (BER) pathway. A more definitive assessment of PARP function was recently provided by the generation of PARP-deficient mice by homologous recombination (35, 53). We found that PARP^−/− mice are hypersensitive to monofunctional alkylating agents and γ-irradiation and display a marked genomic instability (sister chromatid exchanges and chromatid and chromosome breaks) following DNA damage (35). Interestingly, γ-irradiation of these mice causes acute toxicity of the epithelia of their small intestines (35), as has been observed with other DNA damage and signalling and repair enzyme deficiencies (2, 3), thus emphasizing the crucial function of DNA surveillance programs of rapidly dividing cells. Similar results indicating that PARP is important for the maintenance of genomic stability following environmental or experimental stress were recently obtained (54).In this work, we have used the two-hybrid system to identify genes encoding proteins that putatively interact with PARP and are involved in its biological function. The human PARP cDNA fused to the LexA-encoding DNA-binding domain (DBD) was used as bait to screen a HeLa cDNA library fused with the activation domain of Gal4. This screening resulted in the identification of the BER pathway protein XRCC1 (X-ray repair cross-complementing 1) as a factor that associates with PARP. This interaction was further confirmed by in vivo experiments with glutathione S-transferase (GST)-tagged fusion proteins expressed in Cos-7 and HeLa cells. XRCC1 and PARP were found to interact via their respective BRCT (BRCA1 C terminus) modules (4, 9) and via an additional site located in the N-terminal zinc-finger domain of PARP. This association dramatically decreased the catalytic activity of PARP without modifying its nick sensor function. Therefore, the association of PARP with XRCC1, a partner of DNA ligase III (7, 8) and DNA polymerase β (25), is suggestive of a role in the detection and protection of a DNA strand break and the subsequent targeting of a BER complex to the damaged site.     

Gemcitabine Induces Poly (ADP-Ribose) Polymerase-1 (PARP-1) Degradation through Autophagy in Pancreatic Cancer

Poly (ADP-ribose) polymerase-1 (PARP-1) and autophagy play increasingly important roles in DNA damage repair and cell death. Gemcitabine (GEM) remains the first-line chemotherapeutic drug for pancreatic cancer (PC). However, little is known about the relationship between PARP-1 expression and autophagy in response to GEM. Here we demonstrate that GEM induces DNA-damage response and degradation of mono-ADP ribosylated PARP-1 through the autophagy pathway in PC cells, which is rescued by inhibiting autophagy. Hypoxia and serum starvation inhibit autophagic activity due to abrogated GEM-induced mono-ADP-ribosylated PARP-1 degradation. Activation of extracellular regulated protein kinases (ERK) induced by serum starvation shows differences in intracellular localization as well as modulation of autophagy and PARP-1 degradation in GEM-sensitive KLM1 and -resistant KLM1-R cells. Our study has revealed a novel role of autophagy in PARP-1 degradation in response to GEM, and the different impacts of MEK/ERK signaling pathway on autophagy between GEM-sensitive and -resistant PC cells.