A dual approach in the study of poly (ADP-ribose) polymerase: In vitro random mutagenesis and generation of deficient mice (original) (raw)
Regulatory mechanisms of poly(ADP-ribose) polymerase
ADP-Ribosylation Reactions: From Bacterial Pathogenesis to Cancer, 1999
Here, we describe the latest developments on the mechanistic characterization of poly(ADP-ribose) polymerase (PARP) [EC 2.4.2.30], a DNA-dependent enzyme that catalyzes the synthesis of protein-bound ADP-ribose polymers in eucaryotic chromatin. A detailed kinetic analysis of the automodification reaction of PARP in the presence of nicked dsDNA indicates that protein-poly(ADP-ribosyl)ation probably occurs via a sequential mechanism since enzyme-bound ADP-ribose chains are not reaction intermediates. The multiple enzymatic activities catalyzed by PARP (initiation, elongation, branching and self-modification) are the subject of a very complex regulatory mechanism that may involve allosterism. For instance, while the NAD + concentration determines the average ADP-ribose polymer size (polymerization reaction), the frequency of DNA strand breaks determines the total number of ADP-ribose chains synthesized (initiation reaction). A general discussion of some of the mechanisms that regulate these multiple catalytic activities of PARP is presented below. (Mol Cell Biochem 193: 19-22, 1999)
Identification of potential active-site residues in the human poly (ADP-ribose) polymerase
Journal of Biological …, 1993
The carboxyl-terminal catalytic domain of the human poly(ADP-ribose) polymerase (PARP) exhibits sequence homology with the NAD(P)+-dependent leucine and glutamate dehydrogenases. To clarify the role played by some conserved residues between PARP and NAD(P)+-dependent dehydrogenases, point mutations were introduced into the whole enzyme context. Nonconservative mutations of Lys-893 (K893I) and Asp-993 (D993A) completely inactivate human PARP, whereas conservative and nonconservative mutations of Asp-914 (D914E and D914A, respectively) and Lys-953 (K953R and K9531, respectively) partially alter PARP activity. The consequences of conservative substitution of Lys-893 and Asp-993 on the kinetic properties of human poly(ADP-ribose) polymerase enzyme and the polymer it synthesizes suggest that these 2 amino acids are directly involved in the covalent attachment of the first ADP-ribosyl residue from NAD+ onto the acceptor amino acid. In addition, the recent resolution of the three-dimensional structure of the NAD+-linked glutamate dehydrogenase from Clostridium symbiosum ((1992) Proteins 12, 75-86) strongly supports our alignment with leucine and glutamate dehydrogenases and provides an interesting structural framework for the analysis of our results of site-directed mutagenesis, I Belix 10 1 I BE I I EX 1 J Be& 11 1 B G 194 G 944 G 200 G 950 G 201 G 952 G 261 D 993 D 160 D 914 d 237 G 239 G 972 G 974 Frc. 2. Panel A , multiple alignment of the sequence of the carboxyl-terminal region of human PARP (HumPARP) with various Leu/Glu dehydrogenases sequences (Csy GDH, C. symbiosum NAD+ glutamate dehydrogenase; Eco GDH, E. coli NADP+ glutamate dehydrogenase; Bov GDH, bovine NADP+ glutamate dehydrogenase; Yea GDH, Saccharomyces cerevisiae NADP+ glutamate dehydrogenase; Ncr GDH, Neurospora crassa NAD' glutamate dehydrogenase; Bst LDH, Bacillus stearothermophilus NAD+ leucine dehydrogenase). The highly conserved residues indicated in
Journal of Biological Chemistry, 1993
The cDNA encoding the carboxyl-terminal 40-kDa domain of human poly(ADP-ribose) polymerase was inserted into an expression vector. The recombinant protein was overproduced in Escherichia coli, and purified to homogeneity. The 40-kDa domain had the same affinity (ICm) for NAD+ as the full-length enzyme, expressed abortive NAD+ glycohydrolase activity, catalyzed the initiation, elongation, and branching of ADP-ribose polymers, but exhibited no DNA dependence. Its specific activity was approximately 600-fold lower than that of the whole enzyme activated by DNA strand breaks. Surprisingly, the carboxyl-terminal 40- kDa domain exhibited the processive mode of polymer attachment typical of full-length poly(ADP-ribose) polymerase and was able to modify histones H1 and H2B. Finally, the polymer sizes formed by the 40-kDa domain were influenced by histone H1.
Progress in Biophysics and Molecular Biology, 2005
Poly(ADP-ribose) polymerases (PARPs) are a family of enzymes, which show differences in structure, cellular location and functions. However, all these enzymes possess poly(ADP-ribosyl)ation activity. Overactivation of PARP enzymes has been implicated in the pathogenesis of several diseases, including stroke, myocardial infarction, diabetes, shock, neurodegenerative disorder and allergy. The best studied of these enzymes (PARP-1) is involved in the cellular response to DNA damage so that in the event of irreparable DNA damage overactivation of PARP-1 leads to necrotic cell death. Inhibitors of PARP-1 activity in combination with DNA-binding antitumor drugs may constitute a suitable strategy in cancer chemotherapy. In addition, PARP inhibitors may be also useful to restore cellular functions in several pathophysiological states and diseases. This review gives an update of the state-of-the-art concerning PARP enzymes and their exploitation as pharmacological targets in several illnesses.
Expression in E.coli of the catalytic domain of rat poly(ADP-ribose)polymerase
FEBS Letters, 1990
A 2 kilobase pair cDNA coding for the entire C-terminal catalytic domain of rat poly(ADP-riiose)polymerase has been expressed in E. coli. The overproduced 55 kDa polypeptide is active in synthesizing poly(ADP-ribose) and the 4 kDa N-terminal region of this domain is recognized by the monoclonal antibody C I,2 directed against the calf enzyme. Also, the minor a-chymotrypsin cleavage site found in the human catalytic domain is not present in the rat enzyme as revealed by the absence of the 40 kDa specific degradation product in the E. coli cells expressing the rat domain. The expression of this partial rat cDNA should thus permit the rapid purification and subsequent crystallization of the catalytic domain of the enzyme.
Poly(ADP-ribose) polymerase stimulates DNA polymerase alpha by physical association
The Journal of biological chemistry, 1993
The direct effect of the eukaryotic nuclear DNA-binding protein poly(ADP-ribose) polymerase on the activity of DNA polymerase alpha was investigated. Homogenously purified poly(ADP-ribose) polymerase (5 to 10 micrograms/ml) stimulated the activity of immunoaffinity-purified calf or human DNA polymerase alpha by about 6 to 60-fold in a dose-dependent manner. It had no effect on the activities of DNA polymerase beta, DNA polymerase gamma, and primase, indicating that its effect is specific for DNA polymerase alpha. Apparently, poly(ADP-ribosyl)ation of DNA polymerase alpha was not necessary for the stimulation. The stimulatory activity is due to poly(ADP-ribose) polymerase itself since it was immunoprecipitated with a monoclonal antibody directed against poly(ADP-ribose) polymerase. Kinetic analysis showed that, in the presence of poly(ADP-ribose) polymerase, the saturation curve for DNA template primer became sigmoidal; at very low concentrations of DNA, it rather inhibited the react...
Experimental Gerontology, 2000
DNA damage induced in higher eukaryotes by alkylating agents, oxidants or ionising radiation triggers the synthesis of protein-conjugated poly(ADP-ribose) catalysed by poly(ADP-ribose) polymerase-1 (PARP-1). Previously, cellular poly(ADP-ribosyl)ation capacity has been shown to correlate positively with the life span of mammalian species [Proc. Natl. Acad. Sci. USA 89 (1992) 11 759-11 763]. Here, we have tested whether this correlation results from differences in kinetic parameters of the enzymatic activity of PARP-1. We therefore compared recombinant enzymes, expressed in a baculovirus system, from rat and man as two mammalian species with extremely divergent life span. In standard activity assays performed in the presence of histones as poly(ADP-ribose) acceptors both enzymes showed saturation kinetics with [NAD ϩ ]. The kinetic parameters (k cat , k m and k cat /k m ) of the two enzymes were not significantly different. However, in assays assessing the auto-poly(ADP-ribosyl)ation reaction, both enzymes displayed second-order kinetics with respect to , and up to twofold higher specific activity was observed for human versus rat PARP-1. We conclude that the correlation of poly(ADP-ribosyl)ation capacity with life span is not reflected in the kinetic parameters, but that subtle differences in primary structure of PARP-1 from mammalian species of different longevity may control the extent of the automodification reaction. ᭧
Poly(ADP-ribose) polymerase-1: what have we learned from the deficient mouse model?
Mutation research, 2000
Poly (ADP-ribose) polymerase (113 kDa; PARP-1) is a constitutive factor of the DNA damage surveillance network developed by the eukaryotic cell to cope with the numerous environmental and endogenous genotoxic agents. This enzyme recognizes and is activated by DNA strand breaks. This original property plays an essential role in the protection and processing of the DNA ends as they arise in DNA damage that triggers the base excision repair (BER) pathway. The generation, by homologous recombination, of three independent deficient mouse models have confirmed the caretaker function of PARP-1 in mammalian cells under genotoxic stress. Unexpectedly, the knockout strategy has revealed the instrumental role of PARP-1 in cell death after ischemia-reperfusion injury and in various inflammation process. Moreover, the residual PARP activity found in PARP-1 deficient cells has been recently attributed to a novel DNA damage-dependent poly ADP-ribose polymerase (62 kDa; PARP-2), another member of t...