Cancer radiation therapy generates DNA lesions and reactive oxygen species, which activate Poly ADP-ribose polymerase 1 (PARP1) and its related family member PARP2. Activated PARP1/2 transfers ADP- Ribose to themselves and histones. The resulted poly (ADP-ribose) (PAR) chains further recruit others repair proteins (e.g. XRCC1-Lig3 complex). Specific inhibitors for both PARP1 and 2 (refer to as PARPi) preferentially target BRCA1/2 deficient cancer cells and synergize with other genotoxic cancer therapies, including radiation. While these effects were originally attributed to the lack of PAR dependent recruitment of XRCC1, deletion of XRCC1 does not have the same cytotoxicity as PARPi. More recently PARPi was found to trap PARP1 at DNA breaks, converting transient PARP1 foci (<10min) to durable ones (>30 min). If not removed, the PARP1-DNA adduct can prevent DNA repair and stall replication. Accordingly PARP1 deletion ?desensitizes? cancer cells to PARPi, revealing a structural function of PARP1 in cancer therapy. In preliminary studies, we found unexpectedly the release of PARP1 from DNA is regulated by PARP2 and mono-ADP-ribosylation (MARylation), but not PARylation. To understand this structural function of PARP1 in vivo, we generated mouse models expressing constitutive (Parp1A) or inducible (Parp1AN) PARylation defective PARP1 (E988A) and a conditional PARP1 mouse model (Parp1C). In contrast to the normal development and gender distribution of Parp1-/- mice, Parp1+/A mice display female specific embryonic lethality. And male Parp1+/A cells display severe hypersensitivity to DNA damage agents beyond what is found in Parp1-/- ctrl. Given the application of PARP inhibitor in cancer types almost exclusively affecting women (breast and ovarian), this gender bias is intriguing and might be caused by the breaks independent binding of PARP1 on the chromatin and an role of PARP1 protein in X-inactivation and other epigenetic regulation. Female Parp1+/-Parp2-/- (but not Parp1-/-Parp2+/-) mice also display female specific lethality accompanied by X-chromosome specific instability. Based on these findings, we hypothesize that PARP1 has structural function in chromatin biology and DNA repair that are critical for cancer therapy. To test this, we will use mouse genetics, structural biology and cell biology approaches to 1) determine the mechanism that regulates PARP1 dynamics at DNA damage sites, reveal the biological consequence of PARP1 trapping and 2) characterize PARP1 E988A mouse models to determine the impact of catalytic inactive PARP1 on DNA repair, X-inactivation, oncogenesis in vivo. The completion of this study will elucidate the previously un-recognized structural function of PARP1, reveal functional interactions between PARP1 and PARP2 and the gender specific role of PARP1 in female. In the near future, the mechanism and the novel animal models generated in the course of our study will also facilitate the development and use of PARP inhibitors for cancer therapy.
Our proposal utilizes cell biology, molecular biology, structural biology and mouse genetic approaches to identify 1) the mechanism that regulates the disassociation of PARP1 from the DNA damage sites and the impact of trapped PARP1 on DNA repair and DNA damage responses in replicating cells; 2) the effects of PARylation defective PARP1 on epigenetic regulation (e.g. X-inactivation), DNA repair and DNA damage response and tumorigenesis in vivo using animal models. The ultimate goal is to understand the structural function of PARP1 in chromatin biology and DNA repair, and identify genetic pathways and therapeutic interventions (including radiation) that sensitize replicating cells to PARP1 trapping and PARP inhibitors.
