The MDM2 oncoprotein antagonizes Poly(ADP-ribosyl)ation

Abstract

MDM2 is a target gene product and a negative regulator of the tumor suppressor p53. Additionally, MDM2 interferes with the progression of DNA replication forks. Poly(ADP-Ribose) Polymerase 1 (PARP1) is the main ADP-ribosyltransferase in human cells. It regulates DNA replication fork progression and DNA damage tolerance. In this thesis work, we studied the interplay between MDM2 and PARP1, and the functional consequences of it, with a particular focus on DNA replication. We found that p53 activation reduced PARP1 protein levels by the induction of MDM2. MDM2 directly interacts with PARP1, leading to inactivation, ubiquitination and proteasome-mediated degradation of the latter. In the same context, MDM2 significantly enhanced the rate of DNA synthesis. This depended on PARP1 inactivation, since transient overexpression of PARP1 brought DNA replication fork progression back to control levels, and simultaneous MDM2 induction and PARP1 inhibition did not further increase newly synthetized DNA track lengths, strongly suggesting an epistatic relation. When a replication fork encounters an obstacle, fork reversal can occur, meaning that the canonical three-way replication fork is rearranged to a four-way junction. Since PARP1 is required for the stabilization of these structures, we quantified the frequency of reversed forks by electron microscopy. We found that MDM2 accumulation strongly reduced fork reversal. In order to further determine the mechanistic base of the acceleration of DNA replication forks, we investigated the impact of two factors involved in the restart of stalled replication forks, which have been linked to PARP1. These are the helicase RECQ1, which mediates the resolution of reversed replication forks, and the primase/polymerase PRIMPOL, which restarts DNA synthesis downstream replication roadblocks. Strikingly, either RECQ1 or PRIMPOL depletion disabled MDM2-mediated acceleration of the nascent DNA elongation. We then explored whether the enhanced restart at stalled DNA replication forks would provoke genome instability by analyzing the formation of micronuclei. As expected, MDM2-dependent inactivation of PARP1 promoted micronuclei formation. In addition, it also exacerbated the cytotoxicity of the Topoisomerase I inhibitor camptothecin, highlighting a potential clinical relevance of this pathway. In conclusion, MDM2 does not only provide negative feedback on p53; it also antagonizes PARP1 to govern DNA replication fork progression, thus enhancing the tolerance of cells towards DNA damage.