Distribution of Poly(ADP-ribose) Glycohydrolase in Different Functional Domains of the Cell Nucleus

Pacheco-Rodriguez, Gustavo, Distribution of Poly(ADP-ribose) Glycohydrolase in Different Functional Domains of the Cell Nucleus. Doctor of Philosophy (Biomedical Sciences), August, 1996, 147 pp, 3 tables, 44 illustrations, bibliography, 138 titles. In this study, the distributor poly(ADP-ribose) glycohydrolase (PARG) in different subdomains of the cell nucleus and the role of non-covalent interactions of poly(ADP-ribose) with nuclear proteins have been characterized. An assay that allows the simultaneous determination of specific non-covalent interactions of poly (ADP-ribose) with nuclear proteins as well as PARG activity by high resolution polyacrylamide gel electrophoresis was developed. This method was made possible by the enzymatic synthesis of (ADP-ribose)2-70 at 10 μM NAD+ with purified poly(ADP ribose) polymerase (PARP). Either purified or nuclear-associated PARG degraded poly(ADP-ribose) biphasically. Nuclei were fractioned into functional domains namely, chromatin, nuclear matrix and nuclear envelope. These domains were characterized biochemically by their protein composition and by electron microscopy. PARG activity was identified mainly with chromatin and the nuclear matrix. Interestingly, PARG activity was also associated with the nuclear envelope. Thus, the poly(ADP-ribosyl)ation pathway is regulated topologically. It was further determined that poly(ADP-ribose) interacts non-covalently with purified histone proteins or proteins in the nuclear environment. In addition, the nuclear matrix proteins also interacted non-covalently with poly(ADP-ribose). These non-covalent interactions appear to regulate the catabolism of poly(ADP-ribose) via a catabolite intermediate constituted of a [protein][poly(ADP-ribose)] complex. The affinity of the nuclear associated protein responsible for triggering the degradation of poly(ADP-ribose) correlates with the affinity of histone H4 for ADP-ribose chains of 20 residues or more. The findings of this research stresses that : a) poly (ADP-ribose) is catabolized by PARG in vivo; b) PARG is associated with chromatin, nuclear matrix and the nuclear envelope; c) the degradation of poly(ADP-ribose) is dependent on its non-covalent interactions with nuclear proteins; and d) histone H4 appears to be responsible for triggering the catabolism of poly(ADP-ribose).