Rashmi G. Shah

Role of poly(ADP-ribose) polymerase-1 in the removal of UV-induced DNA lesions by nucleotide excision repair

Among the earliest responses of mammalian cells to DNA damage is catalytic activation of a nuclear enzyme poly(ADP-ribose) polymerase-1 (PARP-1). Activated PARP-1 forms the polymers of ADP-ribose (pADPr or PAR) that posttranslationally modify its target proteins, such as PARP-1 and DNA repair–related proteins. Although this metabolism is known to be implicated in other repair pathways, here we show its role in the versatile nucleotide excision repair pathway (NER) that removes a variety of DNA damages including those induced by UV. We show that PARP inhibition or specific depletion of PARP-1 decreases the efficiency of removal of UV-induced DNA damage from human skin fibroblasts or mouse epidermis. Using NER-proficient and -deficient cells and in vitro PARP-1 assays, we show that damaged DNA-binding protein 2 (DDB2), a key lesion recognition protein of the global genomic subpathway of NER (GG-NER), associates with PARP-1 in the vicinity of UV-damaged chromatin, stimulates its catalytic activity, and is modified by pADPr. PARP inhibition abolishes UV-induced interaction of DDB2 with PARP-1 or xeroderma pigmentosum group C (XPC) and also decreases localization of XPC to UV-damaged DNA, which is a key step that leads to downstream events in GG-NER. Thus, PARP-1 collaborates with DDB2 to increase the efficiency of the lesion recognition step of GG-NER.

PARP1 Links CHD2-Mediated Chromatin Expansion and H3.3 Deposition to DNA Repair by Non-homologous End-Joining

Summary The response to DNA double-strand breaks (DSBs) requires alterations in chromatin structure to promote the assembly of repair complexes on broken chromosomes. Non-homologous end-joining (NHEJ) is the dominant DSB repair pathway in human cells, but our understanding of how it operates in chromatin is limited. Here, we define a mechanism that plays a crucial role in regulating NHEJ in chromatin. This mechanism is initiated by DNA damage-associated poly(ADP-ribose) polymerase 1 (PARP1), which recruits the chromatin remodeler CHD2 through a poly(ADP-ribose)-binding domain. CHD2 in turn triggers rapid chromatin expansion and the deposition of histone variant H3.3 at sites of DNA damage. Importantly, we find that PARP1, CHD2, and H3.3 regulate the assembly of NHEJ complexes at broken chromosomes to promote efficient DNA repair. Together, these findings reveal a PARP1-dependent process that couples ATP-dependent chromatin remodeling with histone variant deposition at DSBs to facilitate NHEJ and safeguard genomic stability.

Determination of genotoxicity of the metabolites of the pesticides Guthion, Sencor, Lorox, Reglone, Daconil and Admire by 32P-postlabeling

Commercial formulations of the pesticides: Guthion (azinphos methyl), Sencor (metribuzin), Lorox (linuron), Reglone (diquat), Daconil (chlorothalonil) and Admire (imidacloprid) were studied for their genotoxicity by 32P-postlabeling. Metabolites of the pesticides were obtained enzymatically using arochlor induced rat liver S9 fraction, in an NADPH generating system. The resulting metabolites were reacted with calf thymus DNA and the DNA was analyzed for presence of adducts by either the nuclease P1 or butanol enrichment. Nuclease P1 enrichment resulted in adducts for all the pesticides. Compared to the level of adducts in control DNA, the levels in pesticide-treated DNA were higher for all the pesticides, except Daconil. The increase in adduct numbers for pesticide-treated DNAs ranged from 4.9-12.4 times the control-DNA indicating pesticide genotoxicity in this in vitro system. Enrichment using butanol extraction gave three adducts unique to Sencor-DNA. These adducts were different from those obtained with nuclease P1 enrichment of the same. B(α)P was the positive control for the in vitro metabolism, and two adduct enrichment procedures: nuclease P1 digestion and butanol extraction.

Mechanism of early biphasic activation of poly(ADP-ribose) polymerase-1 in response to ultraviolet B radiation.

The damage to DNA caused by ultraviolet B radiation (280-320 nm) contributes significantly to development of sunlight-induced skin cancers. The susceptibility of mice to ultraviolet B-induced skin carcinogenesis is increased by an inhibitor of the DNA damage-activated nuclear enzyme poly(ADP-ribose) polymerase-1 (PARP), hence PARP activation is likely to be associated with cellular responses that suppress carcinogenesis. To understand the role of activated PARP in these cellular functions, we need to first clearly identify the cause of PARP activation in ultraviolet B-irradiated cells. Ultraviolet B, like ultraviolet C, causes direct DNA damage of cyclobutane pyrimidine dimer and 6, 4-photoproduct types, which are subjected to the nucleotide excision repair. Moreover, ultraviolet B also causes oxidative DNA damage, which is subjected to base excision repair. To identify which of these two types of DNA damage activates PARP, we examined mechanism of early PARP activation in mouse fibroblasts exposed to ultraviolet B and C radiations. The ultraviolet B-irradiated cells rapidly activated PARP in two distinct phases, initially within the first 5 minutes and later between 60-120 minutes, whereas ultraviolet C-irradiated cells showed only the immediate PARP activation. Using antioxidants, local irradiation, chromatin immunoprecipitation and in vitro PARP assays, we identified that ultraviolet radiation-induced direct DNA damage, such as thymine dimers, cause the initial PARP activation, whereas ultraviolet B-induced oxidative damage cause the second PARP activation. Our results suggest that cells can selectively activate PARP for participation in different cellular responses associated with different DNA lesions.

IMMUNOLOGICAL DETERMINATION AND SIZE CHARACTERIZATION OF POLY(ADP-RIBOSE) SYNTHESIZED IN VITRO AND IN VIVO

Poly(ADP-ribose) polymerase is a DNA break detecting enzyme playing a role in the surveillance of genome integrity. Poly(ADP-ribose) is synthesized rapidly and transiently from beta-NAD in response to DNA damaging agents. In order to study the physiological significance of poly(ADP-ribose) metabolism, we have developed immunological methods which enable us to study endogenous poly(ADP-ribose) without interfering with cell metabolism and integrity. For this purpose, we produced a highly specific polyclonal anti-poly(ADP-ribose) antibody which immunoreacts with polymers and oligomers. In addition to the immunodot blot method recently described by us (Affar et al., Anal. Biochem. 259 (1998) 280-283), other applications were investigated in cells: (i) detection of poly(ADP-ribose) by ELISA; (ii) characterization of poly(ADP-ribose) size using high resolution gel electrophoresis of polymers, followed by its transfer onto a positively charged membrane and detection with anti-poly(ADP-ribose) antibody; (iii) immunocytochemistry and flow cytometry analyses allowing poly(ADP-ribose) study at the level of individual cells.

Biochemical Assessment of Niacin Deficiency Among Carcinoid Cancer Patients

OBJECTIVE:Carcinoid cancer patients often have elevated levels of serotonin or its precursor 5-hydroxytryptophan. Normally, serotonin synthesis accounts for a small fraction of tryptophan catabolism, which should be directed along a pathway that allows partial conversion to niacin; hence, increased diversion of tryptophan toward serotonin could cause variable degrees of niacin deficiency in carcinoid patients. Therefore, the prevalence of niacin deficiency among carcinoid patients was investigated by clinical assessment of pellagra and biochemical assessment of whole blood niacin number, a ratio derived from two biologically active forms of niacin (NAD/NADP × 100).METHODS:Clinical and biochemical niacin status were assessed in a cohort of newly diagnosed carcinoid patients with carcinoid syndrome (CCS, n = 36), carcinoid patients without carcinoid syndrome (CWCS, n = 32) and noncarcinoid controls (n = 24) recruited at two primary care clinics. Other aspects of serotonin metabolism were measured by analyses of plasma serotonin and tryptophan and urinary excretion of 5-hydroxyindoleacetic acid.RESULTS:Biochemical niacin deficiency (niacin number < 130) was significantly more common in CCS patients (10 out of 36) compared to controls (p < 0.05, Fishers exact test), while CWCS patients displayed an incidence that was not significantly elevated (4 out of 32). Only one CCS patient, who was also identified biochemically as niacin deficient, was clinically diagnosed with pellagra.CONCLUSION:Biochemical niacin deficiency is more prevalent among newly diagnosed CCS patients than in controls. Manifestation of pellagra is a less sensitive indicator, and dependence on this endpoint could lead to a lack of appropriate nutritional support for this group of patients.

Role of poly(ADP-ribose) polymerase in rapid intracellular acidification induced by alkylating DNA damage

In response to high levels of DNA damage, catalytic activation of the nuclear enzyme poly(ADP-ribose) polymerase (PARP) triggers necrotic death because of rapid consumption of its substrate β-nicotinamide adenine dinucleotide and consequent depletion of ATP. We examined whether there are other consequences of PARP activation that could contribute to cell death. Here, we show that PARP activation reaction in vitro becomes acidic with release of protons during hydrolysis of β-nicotinamide adenine dinucleotide. In the cellular context, we show that Molt 3 cells respond to DNA damage by the alkylating agent N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) with a dose-dependent acidification within 30 min. Whereas acidification by 0.15 pH units induced by 10 μM MNNG is reversed within 1 h, 100 μM MNNG-induced acidification by 0.5–0.6 pH units is persistent up to 7 h. Acidification is a general DNA damage response because H2O2 exposure also acidifies Molt 3 cells, and MNNG causes acidification in Jurkat, U937, or HL-60 leukemia cells and in PARP+/+ fibroblasts. Acidification is significantly decreased in the presence of PARP inhibitors or in PARP−/− fibroblasts, suggesting a major role for PARP activation in acidification. Inhibition of proton export through ATP-dependent Na+/H+ exchanger is another major cause of acidification. Using the pH clamp method to either suppress or introduce changes in cellular pH, we show that brief acidification by 0.5–0.6 pH units may be a negative regulator of apoptosis while permitting necrotic death of cells with extensively damaged DNA.

Analysis of ADP-ribose polymer sizes in intact cells.

Poly(ADP-ribose) is a polymer (pADPr) that is synthesized by poly (ADP-ribose) polymerases in response to DNA damaging agents. For instance, chemical alkylating agents such as MNNG [1] or physical stimulation of cells by γ-rays [2] are well known to induce pADPr synthesis. PARPs are members of a growing family of enzymes which includes PARP-1, PARP-2, S-PARP-1, tankyrase and V-PARP [3]. The association of PARP-1 and PARP-2 in DNA damage signaling pathways has been characterized, but tankyrase and V-PARP seem to be independent of DNA repair mechanisms.Poly(ADP-ribosyl)ation leads to heterogenous chain lengths of up to 200 units (mers) in vitro [3]. While most of these will be covalently bound to proteins, they may be released under alkaline conditions for analysis. Previous immunological methods such as immunoblots [4] showed that about 60–70% of the 6–8 mers pADPr were lost during fixation and that the very short pADPr (2–5 mers) were very weakly bound to the membrane [5]. Furthermore, detection of cellular pADPr using enzyme-linked immunosorbent assay (ELISA) revealed that some molecules of pADPr are also lost during fixation and washings. This phenomenon leads to underestimation of the short pADPr population in cells. Thus, evaluating which pADPr sizes are present in cells and tissues becomes critical.We report here the development of a new highly sensitive immunological method to detect synthesized pADPr sizes distribution in intact cells.

Approaches to Detect PARP-1 Activation In Vivo, In Situ, and In Vitro

An accurate and sensitive detection of catalytic activation of poly(ADP-ribose) polymerase-1 (PARP-1) is required to be performed in a wide variety of samples because this activity plays a role in various cellular responses to DNA damage ranging from DNA repair to cell death, as well as in housekeeping functions, such as transcription. Since PARP-1 gene is expressed constitutively, its activation cannot be surmised from increased expression of its mRNA or protein, but by demonstrating the consequences of its catalytic -reaction which results in consumption of the substrate nicotinamide adenine dinucleotide (NAD(+)) and formation of three products, namely, polymer of ADP-ribose (pADPr or PAR), nicotinamide, and protons. Here, we describe various approaches commonly used in our laboratory for detection of PARP-1 activation in vivo (cells, tissues, and tumors), in situ, and in vitro via assessment of formation of pADPr, depletion of the substrate NAD, or formation of protons resulting in rapid and reversible intracellular acidification. It is important to note that although some other members of the PARP family can carry out the same catalytic reaction, many of these assays largely reflect PARP-1 activation in a vast majority of the experimental circumstances and more specifically in DNA damage responses. However, if required, PARP-1-specific action should be confirmed by use of PARP-1 knockout or RNAi-mediated knockdown approaches.

Poly(ADP-ribose) polymerase 1 escorts XPC to UV-induced DNA lesions during nucleotide excision repair

Significance Repair of the majority of UV-induced DNA damage in mammalian cells by the nucleotide excision repair pathway starts with rapid recruitment of Xeroderma pigmentosum C (XPC) protein to the lesion. However, rapidity of XPC recruitment to the lesion site in a genomic context cannot be fully explained by the known properties of XPC or its partner protein DDB2. Here, we show that the DNA damage-detecting nuclear protein PARP1 forms a stable complex with XPC before DNA damage and transfers it very rapidly to the DNA lesion site if other repair conditions are present. Since PARP1 is known to interact with many proteins under steady-state conditions, our results reveal a paradigm that an association with PARP1 could confer a functional advantage to these proteins. Xeroderma pigmentosum C (XPC) protein initiates the global genomic subpathway of nucleotide excision repair (GG-NER) for removal of UV-induced direct photolesions from genomic DNA. The XPC has an inherent capacity to identify and stabilize at the DNA lesion sites, and this function is facilitated in the genomic context by UV-damaged DNA-binding protein 2 (DDB2), which is part of a multiprotein UV–DDB ubiquitin ligase complex. The nuclear enzyme poly(ADP-ribose) polymerase 1 (PARP1) has been shown to facilitate the lesion recognition step of GG-NER via its interaction with DDB2 at the lesion site. Here, we show that PARP1 plays an additional DDB2-independent direct role in recruitment and stabilization of XPC at the UV-induced DNA lesions to promote GG-NER. It forms a stable complex with XPC in the nucleoplasm under steady-state conditions before irradiation and rapidly escorts it to the damaged DNA after UV irradiation in a DDB2-independent manner. The catalytic activity of PARP1 is not required for the initial complex formation with XPC in the nucleoplasm but it enhances the recruitment of XPC to the DNA lesion site after irradiation. Using purified proteins, we also show that the PARP1–XPC complex facilitates the handover of XPC to the UV-lesion site in the presence of the UV–DDB ligase complex. Thus, the lesion search function of XPC in the genomic context is controlled by XPC itself, DDB2, and PARP1. Our results reveal a paradigm that the known interaction of many proteins with PARP1 under steady-state conditions could have functional significance for these proteins.