Abstract

A set of gene constructed encoding the wild-type PARP, the various combinations of the individual domains of PARP, and several mutations of the individual domains have been obtained. All these genes are overexpressed in E. coli, and the purified proteins are used in different combinations to assess their competence as catalyst or as acceptors in poly(ADP-ribosyl)ation reactions. One truncated PARP mutant showed no self-modification activity and no catalytic activities when histone or PARP automodification domain are used as substrates. However, this mutant is able to use all PARP fragments that contain intact DNA-binding domain as substrates for poly(ADP-ribosyl)ation reactions. These results not only reveal the importance of molecular recognition between PARP and its targets, but also indicate the existence of additional modification sites at the C-terminal of PARP DNA binding domain.To gain further insight into the modification sites on PARP and its substrate proteins, and to learn more about the properties of the ADP-ribose polymer, we also synthesized several fluorinated NAD analogues and applied them to poly(ADP-ribosyl)ation reactions. One of these analogues, 2’-deoxy-2’-F-NAD+ in which the 2’ hydroxyl of nicotinamide ribose of NAD+ is substituted by a fluoro atom, should inhibit the branching reaction of PARP. As expected, our results indicate that with this analogue as co-substrate, PARP modifies the substrate proteins by forming non-branched polymers which can be recognized by anti-PAR antibody. This analogue provides us an easy way to distinguish types of modified proteins and will be useful in searching for PARP substrates. These studies and results obtained from our ongoing research with the NAD+ analogues in biological applications will be presented. References: 1.Griesenbeck J, Oei SL, Mayer-Kuckuk P, Ziegler M, Buchlow G, Schweiger M: Biochemistry, 1997; 36: 7297 2.de Murcia G, Shall S: From DNA damage and stress signaling to cell death polyADP-ribosylation reactions. Oxford University Press, New York 2000, 35-79 3.Ruf A, Rolli V, de Murcia G, Schulz GE: J Mol Biol, 1998; 278: 57-65

Keywords: truncated PARP, fluorinated NAD+ analogues