Roles of poly(ADP-ribosyl)ation and PARP in apoptosis, DNA repair, genomic stability and functions of p53 and E2F-1

In this chapter, a variety of different techniques were employed, focusing on various roles for the poly(ADP-ribosyl)ation of nuclear proteins. Data presented here on apoptosis demonstrated that PARP not only plays a passive role by being cleaved during apoptosis, but that under certain circumstances, early, still-to-be-discovered events of apoptosis require brief and transient poly(ADP-ribosyl)ation reactions. One protein which may is modified is the tumor suppressor p53. Data were presented indicating that when this molecule was poly(ADP-ribosyl)ated during apoptosis, it is highly negatively charged and cannot activate p53-responsive genes. However, when PAR is enzymatically removed from this protein later in apoptosis, these genes are induced. Direct evidence for this was provided by gel shift analysis. Thus, a cycling mechanism for poly(ADP-ribosyl)ated proteins is suggested by our data, and direct AFM data was presented, which physically showed such a cycling mechanism for PARP. Clearly the PARP-K/O animals have proven very valuable to help clarify the various pleiotropic roles that PARP plays in cells. Genetic analysis, including CGH, showed that the absence of PARP causes genomic instability in cells, resulting in gains and losses in three specific chromosomes in the mouse genome. Much more data needs to be accumulated to determine which genes have been gained or lost due to PARP deficiency, but our data thus far indicates that the PARP K/O mice have, at a minimum, lost one tumor suppressor (Rb) and have gained an oncogene, Jun. Clearly, much more information is required to understand this complex protein modification - which presumably plays a number of roles in cells, such as replication, repair, and apoptosis. However, the molecular techniques that our laboratory, as well as others, have utilized has begun in recent years to elucidate potential roles for this very interesting and abundant nuclear enzyme.