A dual approach in the study of poly (ADP-ribose) polymerase: In vitro random mutagenesis and generation of deficient mice (original) (raw)
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Requirement of poly(ADP-ribose) polymerase in recovery from DNA damage in mice and in cells
Proceedings of the National Academy of Sciences, 1997
Poly(ADP-ribose) polymerase [PARP; NAD + ADP-ribosyltransferase; NAD + : poly(adenosine-diphosphate- d -ribosyl)-acceptor ADP- d -ribosyltransferase, EC 2.4.2.30 ] is a zinc-finger DNA-binding protein that detects specifically DNA strand breaks generated by genotoxic agents. To determine its biological function, we have inactivated both alleles by gene targeting in mice. Treatment of PARP −/− mice either by the alkylating agent N -methyl- N -nitrosourea (MNU) or by γ-irradiation revealed an extreme sensitivity and a high genomic instability to both agents. Following whole body γ-irradiation (8 Gy) mutant mice died rapidly from acute radiation toxicity to the small intestine. Mice-derived PARP −/− cells displayed a high sensitivity to MNU exposure: a G 2 /M arrest in mouse embryonic fibroblasts and a rapid apoptotic response and a p53 accumulation were observed in splenocytes. Altogether these results demonstrate that PARP is a survival factor playing an essential and positive role d...
European Journal of Biochemistry, 1997
Poly(ADP-ribosy1)ation is a posttranslational modification of nuclear proteins catalyzed by poly(ADPribose) polymerase (PARP), an enzyme which uses NAD' as substrate. Binding of PARP to DNA singlestrand or double-strand breaks leads to enzyme activation. Inhibition of poly(ADP-ribose) formation impairs the cellular recovery from DNA damage. Here we describe stable transfectants of the Chinese hamster cell line C060 that constitutively overexpress human PARP (COCF clones). Immunofluorescence analysis of y-irradiation-stimulated poly(ADP-ribose) synthesis revealed consistently larger fractions of cells positive for this polymer in the COCF clones than in control clones, which failed to express human PARP. HPLC-based quantitative determination of in vivo levels of poly(ADP-ribose) confirmed this result and revealed that the basal polymer levels of undamaged cells were significantly higher in the COCF clones. The COCF clones were sensitized to the cytotoxic effects of y irradiation compared with control transfectants and parental cells. This effect could not be explained by depletion of cellular NAD' or ATP pools. Together with the well-known cellular sensitization by inhibition of poly(ADP-ribosyl)ation, our data lead us to hypothesize that an optimal level of cellular poly(ADP-ribose) accumulation exists for the cellular recovery from DNA damage.
Mutation research, 1994
Poly(ADP-ribose)polymerase (PARP) is a DNA-binding protein that is activated upon induction of DNA breaks and supposed to play a role in DNA repair. To elucidate the effect of overexpression of PARP on the resistance of cells to mutagens, Chinese hamster ovary cells (both the line CHO-9 and the mutagen-hypersensitive derivative 27-1) were transfected with the human PARP cDNA along with pSV2neo. Treatment of the transfected cell population with a high dose of MNNG and selection with G418 gave rise to a significant increase of neo+ clones, as compared to the control transfection with pSV2neo + salmon sperm DNA. The frequency of survivors in these mass culture experiments was lower, however, than after transfection with the bacterial ada gene encoding the DNA repair protein O6-alkylguanine-DNA alkyltransferase. Thus transfection of PARP cDNA in CHO cells is only weakly effective in inducing alkylation resistance. This was confirmed by analyzing the mutagen resistance of individual PARP...
Radiation Oncology Investigations, 1993
Poly(adenosine diphosphate ribose) [poly(ADP-ribose)] polymerase is a mammalian enzyme which synthesizes long chains of ADP-ribose attached to various nuclear proteins in response to DNA strand breaks. A role for this enzyme in cellular radioresistance has been postulated due to the radiosensitizing effect of chemical inhibitors of the enzyme on some cell lines. Inhibitor studies, however, lack specificity and direct evidence for involvement of the enzyme in radioresistance is still needed. In experiments described here, intracellular levels of the enzyme were modulated using vectors to express sense and antisense human cDNA transcripts of poly(ADP-ribose) polymerase in stably transfected NIH/3T3 mouse fibroblasts. Although antisense constructs failed to lower enzyme levels, sense transcripts increased enzyme levels. None of the transfectants, however, showed any difference in either radiation sensitivity or DNA strand break repair rates. These results suggest that simple elevation of poly(ADP-ribose) polymerase does not affect the intrinsic cellular radioresistance of these cells. Rudiut Oncol Invest I993;l :I 89-197. 0 1993 Wiley-Liss, Inc.
Dna Repair, 2005
Poly(ADP-ribosyl)ation is one of the first responses to DNA damage in mammals. Although it is involved in base excision repair, its exact role has not been ascertained yet. Poly(ADP-ribose) polymerase-1 (PARP-1) and PARP-2 mediate most of the poly(ADP-ribosyl)ation response in mammals and are well conserved in evolution. Their respective homologues PME-1 and PME-2 are found in the nematode Caenorhabditis elegans, a well-known genetically tractable model currently used in DNA damage response research. Here we report the functional analysis of PME-1 and PME-2 in presence of DNA damage. Worms irradiated with high doses of ionizing radiations displayed a sharp drop in their NAD + content immediately after treatment, and a biphasic increase in poly(ADP-ribose). The physiological importance of the poly(ADP-ribosyl)ation response was highlighted when worms were preincubated with mammalian PARP inhibitors (3AB, DHQ, PJ34) and irradiated. The embryonic survival rate of the progeny was significantly decreased in a dose-dependent manner. The inhibitor 3AB had a weak effect on embryonic survival, followed closely by DHQ. However, PJ34, a member of the phenantridinone family, was very effective even when used at low concentration (100 nM). In vitro PARP assay using recombinant PME-1 and PME-2 showed a similar pattern of inhibition where 3AB and DHQ were weak inhibitors, and PJ34 a stronger one. Inhibitors affect mostly the poly(ADP-ribose) polymers elongation at high concentrations. These results suggest that poly(ADP-ribosyl)ation in response to DNA damage is an ancient and very important biochemical process protecting DNA from deleterious modification.
Poly (ADP-ribose) polymerase(PARP-1) is not involved in DNA double- …
BMC Cell …
PARP-1 -/- were considerably more sensitive than PARP-1 +/+ 3T3s to induced cell kill by γ-rays and H 2 O 2 . However, the two cell lines did not show any significant difference in the susceptibility to neocarzinostatin below 1.5 nM drug. Restoration of PARP-1 expression in PARP-1 -/- ...
PARP-2, A Novel Mammalian DNA Damage-dependent Poly(ADP-ribose) Polymerase
Journal of Biological Chemistry, 1999
Poly(ADP-ribosylation) is a post-translational modification of nuclear proteins in response to DNA damage that activates the base excision repair machinery. Poly-(ADP-ribose) polymerase which we will now call PARP-1, has been the only known enzyme of this type for over 30 years. Here, we describe a cDNA encoding a 62-kDa protein that shares considerable homology with the catalytic domain of PARP-1 and also contains a basic DNA-binding domain. We propose to call this enzyme poly(ADP-ribose) polymerase 2 (PARP-2). The PARP-2 gene maps to chromosome 14C1 and 14q11.2 in mouse and human, respectively. Purified recombinant mouse PARP-2 is a damaged DNA-binding protein in vitro and catalyzes the formation of poly(ADP-ribose) polymers in a DNA-dependent manner. PARP-2 displays automodification properties similar to PARP-1. The protein is localized in the nucleus in vivo and may account for the residual poly(ADP-ribose) synthesis observed in PARP-1-deficient cells, treated with alkylating agents or hydrogen peroxide.
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...
2003
DNA-dependent protein kinase (DNA-PK) and poly(ADP-ribose) polymerase-1 (PARP-1) participate in non-homologous end joining and base excision repair respectively, and are key determinants of radio-resistance. The interactive effects of PARP-1 and DNA-PK in the cellular responses to DNA damage were investigated using novel specific inhibitors of DNA-PK (NU7026) and PARP-1 (AG14361) and cell lines proficient or deficient for DNA-PK or PARP-1. Enzyme deficient cell lines were 4-fold more sensitive to ionizing radiation (IR) alone, and showed reduced potentially lethal damage recovery (PLDR), compared to their proficient counterparts. NU7026 potentiated IR cytotoxicity in exponentially growing DNA-PK proficient, but not deficient cells. Similarly AG14361 potentiated IR in PARP-1 +/+ but not PARP-1-/cells. When NU7026 and AG14361 were used in combination, their potentiating effects were additive. Both inhibitors reduced PLDR in the proficient cell lines. Furthermore, inhibitor combination...