Abstract

Poly(ADP-ribose) polymerase-1 (PARP-1) is an abundant DNA damage response protein. The binding of PARP-1 to DNA breaks through two zinc finger motifs in the N-terminal domain (24-kDa) activates its C-terminal catalytic domain, which polymerizes ADP-ribose chains onto various nuclear proteins, including itself, which ironically leads to its inactivation and dissociation from the breaks. This DNA break binding and dissociation activity of PARP-1 was thought to be key to DNA repair during DNA damage responses. However, it has been shown that PARP-1 is not essential for the process of repair per se . Conversely, accumulating data point to a pivotal role of PARP-1 in transcription regulation. In this work, we first demonstrated a novel transcription regulation pathway in response to DNA damage by the binding of PARP-1 to RNA secondary structures via the 24-kDa. This high affinity binding was shown to suppress the step of transcription elongation under normal conditions, while automodification of PARP-1 in response to DNA damage causes the release of this inhibition. Paradoxically, PARP-1 has also been shown to promote transcription initiation in undamaged cells by interacting with various transcription factors. Similarly, in the second part of this thesis, we demonstrated a functional interaction between PARP-1 and topoisomerase I (topo I), another abundant enzyme crucial to the process of transcription. We showed that the two proteins show a quasi-identical subnuclear localization throughout the cell cycle and that the specific activity of topo I is increased by physical interaction with PARP-1. Furthermore, we found that PARP-1 deficient cells have lower topo I activities than wild-type cells, a phenotype rescued by the transfection of PARP-1. In the last part of this work, we illustrated how, during apoptosis, cells inhibit transcription, and other futile processes at this stage, such as DNA repair, by separating the 24-kDa fragment from the catalytic domain by proteolytic digestion. This cleavage enhances apoptosis by allowing the 24-kDa fragment to irreversibly bind to RNA and DNA breaks and inhibit transcription and repair, respectively. Taken together, we propose that PARP-1 plays the role of a central DNA damage-dependent transcription coordinator by anchoring itself onto RNA, keeping transcription elongation in check, while interacting with selective factors to promote transcription initiation. Once the cell commits to apoptosis, PARP-1 is cleaved such that the 24-kDa can inhibit transcription and DNA repair, in order for the cell to concentrate its energy for the dying process.