We have studied the apoptotic response of poly(ADPribose) polymerase (PARP)؊/؊ cells to different inducers and the consequences of the expression of an uncleavable mutant of PARP on the apoptotic process. The absence of PARP drastically increases the sensitivity of primary bone marrow PARP؊/؊ cells to apoptosis induced by an alkylating agent but not by a topoisomerase I inhibitor CPT-11 or by interleukin-3 removal. cDNA of wild type or of an uncleavable PARP mutant (D214A-PARP) has been introduced into PARP؊/؊ fibroblasts, which were exposed to anti-CD95 or an alkylating agent to induce apoptosis. The expression of D214A-PARP results in a significant delay of cell death upon CD95 stimulation. Morphological analysis shows a retarded cell shrinkage and nuclear condensation. Upon treatment with an alkylating agent, expression of wild-type PARP cDNA into PARP-deficient mouse embryonic fibroblasts results in the restoration of the cell viability, and the D214A-PARP mutant had no further effect on cell recovery. In conclusion, PARP؊/؊ cells are extremely sensitive to apoptosis induced by triggers (like alkylating agents), which activates the base excision repair pathway of DNA, and the cleavage of PARP during apoptosis facilitates cellular disassembly and ensures the completion and irreversibility of the process.Apoptosis or programmed cell death is a fundamental biological process that plays an important role in early development, cell homeostasis, and in diseases such as neurodegenerative disorders and cancer (1-3). Programmed cell death can occur in response to many stimuli such as genotoxic insult when DNA repair is saturated, removal of growth factors, or activation of the CD95 antigen by CD95 ligand or anti-CD95 antibodies. Morphologically it is characterized by the appearance of membrane blebbing, cell shrinkage, chromatin condensation, and DNA cleavage, and finally the cell is fragmented into membrane-bound apoptotic bodies. At the biochemical level, there is increasing evidence for a central role of the family of cysteine proteases, the caspases, in the pathway that mediates the highly ordered process leading to cell death (4). Caspases have been identified as the enzymes responsible for the proteolysis of key proteins to be selectively cleaved at the onset of apoptosis. It appears that the role of these proteases in cell suicide is to disable critical homeostatic and repair enzymes as well as key structural components. A discrete but increasing number of specific proteins appears to be targeted for proteolytic cleavage during apoptosis, including poly(ADP-ribose) polymerase (PARP, 1 EC 2.4.2.30), which was first described in Ref. 5. In the last years, cleavage of PARP has been used extensively as a marker of apoptosis. However, the reason for the cell to inactivate this protein during the execution phase of apoptosis is not fully understood.PARP is a nuclear zinc finger DNA-binding protein that detects and binds to DNA strand breaks. PARP has a modular organization comprising a NH 2 -terminal DNA-binding dom...

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.In response to DNA-strand breaks introduced either directly by ionizing radiation or indirectly following enzymatic incision at a DNA lesion, the immediate poly(ADP-ribosylation) of nuclear proteins converts the DNA ends into intracellular signals that modulate DNA repair and cell survival programs. At the sites of DNA breakage, poly(ADP-ribose) polymerase (PARP) 1 (EC 2.4.2.30) catalyzes the transfer of the ADP-ribose moiety from its substrate NAD ϩ , to a limited number of proteins involved in chromatin architecture, DNA repair, or in DNA metabolism including PARP itself (1-4). Recently, the generation of PARP-deficient mice by homologous recombination (5, 6) has clearly demonstrated the involvement of PARP in the maintenance of the genomic integrity due to its role during base excision repair (7-9). An substantial delay in DNA strandbreak repair was observed following treatment of PARP-deficient cells with monofunctional alkylating agents (10). This severe DNA repair defect appears to be the primary cause for the observed cytotoxicity of N-methyl-N-nitrosourea, methylmethanesulfonate (MMS), or ␥-rays leading to cell death occurring after a G 2 /M block (10).It was assumed for many years that PARP activity was associated with a single protein displaying unique DNA damage detection and signaling properties. This assumption was challenged by the recent discovery in Arabidopsis thaliana of a gene coding for a PARP-related polypeptide of a calculated molecular mass of 72 kDa (11). It then became evident that two structurally different PARP proteins, both possessing DNA-dependent poly(ADP-ribose) activities, were present in both A. thaliana as well as in maize (12)(13)(14). 2 Furthermore, it has been reported recently that mouse embryonic fibroblasts derived from PARP knockout are capable of synthesizing ADP-ribose polymers in response to DNA damage (15), suggesting that in mammals, like in plants, at least one additional member of the PARP family may exist in addition to the classical zinc finger containing PAR...

We have established a protocol allowing transient and inducible coexpression of many foreign genes in Drosophila S2 Schneider cells. With this powerful approach of reverse genetics, we studied the interaction of the protein tyrosine kinases Syk and Lyn with the B cell antigen receptor (BCR). We find that Lyn phosphorylates only the first tyrosine whereas Syk phosphorylates both tyrosines of the BCR immunoreceptor tyrosine-based activation motif (ITAM). Furthermore, we show that Syk is a positive allosteric enzyme, which is strongly activated by the binding to the phosphorylated ITAM tyrosines, thus initiating a positive feedback loop at the receptor. The BCR-dependent Syk activation and signal amplification is efficiently counterbalanced by protein tyrosine phosphatases, the activity of which is regulated by H(2)O(2) and the redox equilibrium inside the cell.