Abstract
Most of the studies concerning the role of poly(ADP-ribosyl)ation in cellular physiology relied on NAD analogs, e.g. benzamide and derivatives, as competitive PARP* inhibitors. Since such drugs have side-effects on other cellular functions we decided to overexpress selectively the PARP DNA-binding domain as a dominant-negative mutant of for inhibiting this enzyme activity in a highly specific manner (Küpper et al., 1990). Here we review the construction of eucaryotic expression plasmids carrying the PARP full-length open reading frame and a truncated cDNA coding for the DNA-binding domain, respectively. Transfection of these constructs into eucarytic cell lines and monitoring PARP activity in transfected cells clearly showed enhanced enzyme activity in the case of overexpression of the full-length open reading frame. By contrast, transfection of plasmids coding for the DNA-binding domain resulted in a drastic inhibition of poly(ADP-ribosyl)ation, as predicted (Küpper et al., 1990).
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The abbreviations used are: FITC, fluorescein isothiocyanate; HEF, human embryonic fibroblasts; MNNG, N-methyl-N’-nitro-N-nitrosoguanidine; PARP, poly(ADP-ribose) polymerase; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; SV40, Simian virus 40; TCA, trichloro acetic acid; TRITC, tetramethylrhodamine isothiocyanate.
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Küpper, JH., Bürkle, A. (1992). Expression of the DNA-Binding Domain of Human Poly(ADP-Ribose) Polymerase as a Trans-Dominant Inhibitor of Poly(ADP-Ribosyl)ation in Transfected Eucaryotic Cell Lines. In: Poirier, G.G., Moreau, P. (eds) ADP-Ribosylation Reactions. Springer, New York, NY. https://doi.org/10.1007/978-1-4419-8718-1_5
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DOI: https://doi.org/10.1007/978-1-4419-8718-1_5
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